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Impact of the Environment on the PNIPAM Dynamical Transition Probed by Elastic Neutron Scattering
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  • Benedetta P. Rosi
    Benedetta P. Rosi
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    Arianna D’Angelo
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    Jülich Centre for Neutron Science at Heinz Maier-Leibnitz Zentrum, Forschungszentrum Jülich GmbH, Lichtenbergstrasse 1, 85747 Garching, Germany
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    Dipartimento di Fisica e Geologia, Università di Perugia, Via Alessandro Pascoli, 06123 Perugia, Italy
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Macromolecules

Cite this: Macromolecules 2022, 55, 11, 4752–4765
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https://doi.org/10.1021/acs.macromol.2c00177
Published June 1, 2022

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Abstract

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By means of elastic incoherent neutron scattering, we investigated how the addition of stabilizing cosolvents (glycerol and glucose) affects the dynamics of hydrated PNIPAM chains at the pico- and nanosecond time scale, where a low-temperature dynamical transition is observed. From the elastic intensities, the atomic mean square displacements of the PNIPAM samples were extracted using a global fitting procedure. Both the dynamical transition temperature Td and the amplitude of the displacements are found to be strongly dependent on solvent composition. The close analogies between the dynamical transition of PNIPAM and that of biomolecules reveal PNIPAM as an excellent system for reproducing complex solvent–biopolymer interactions.

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1. Introduction

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Smart responsive polymers are a subject of strong interest within the scientific community due to their ability to undergo drastic changes in response to variations of external parameters, such as temperature, pH, or solvent composition. (1) A paradigmatic example of smart polymers is poly-N-isopropylacrylamide (PNIPAM), whose repeating unit includes both hydrophilic amide and hydrophobic isopropyl groups (Scheme 1). The resulting amphiphilic nature produces inverse solubility in water upon heating and triggers a coil-to-globule transition of the polymer at a lower critical solution temperature (LCST) of about 305 K. (2) The LCST located between room and body temperature makes PNIPAM versatile for biological and biomedical applications, ranging from tissue engineering to drug delivery. (3−6)

Scheme 1

Scheme 1. Chemical Formula of the NIPAM Repeating Unita
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aThe C atoms of the amide (hydrophilic) and isopropyl (hydrophobic) groups in the side chain are highlighted in blue and green, respectively. The exchangeable H atom in the amide group is highlighted in red.

In a similar context of biophysical interest, PNIPAM has raised further attention as a simple but predictive protein model, since the coil-to-globule transition in many aspects resembles the cold renaturation of small globular proteins. (7−14) From a fundamental point of view, the availability of such a model system would provide unique opportunities to reproduce, better understand, and fine tune the essential elements at the basis of complex biological mechanisms. From a technological point of view, the perspective of functionalizing PNIPAM to replicate specific biological processes may have a huge impact, for instance, on large-scale applications and analysis. Indeed, the extensive use of natural enzymes is often limited by high costs, demanding storage conditions, and poor availability due to the usually low yield of protein expression. These shortcomings could be overcome by exploiting the higher chemical stability and malleability, abundance, and cheapness of PNIPAM-based systems. (15−17)
Although interest in the coil-to-globule transition has produced vast literature on the behavior of PNIPAM solutions in diluted regime and around room temperature, (18) other regions of the phase diagram are largely unexplored. Only recently, neutron scattering experiments and molecular dynamics simulations disclosed the existence of a dynamical transition in weakly hydrated PNIPAM samples at a critical temperature Td ≈ 220 K. (19−21) Originally observed in proteins (22,23) and later in DNA (24,25) and t-RNA, (26,27) the dynamical transition is a well-known phenomenon in the physics of hydrated biomolecules, whose origin is still widely debated. (28−30) It appears as a slope change at Td in the temperature dependence of the atomic mean square displacements (MSDs) of the macromolecule. Above Td, activation of conformational degrees of freedom takes place, resulting in a faster increase with temperature of the MSDs on the picosecond and nanosecond time scales and a correspondingly enhanced molecular flexibility. Common features of the transition, manifested by both biopolymers and PNIPAM, are (i) the intimate coupling between solvent and solute dynamics, attested by the occurrence of an analogous dynamical transition in the hydration water of the macromolecule (20,31) and by the progressive disappearance of the transition upon dehydration, and (ii) the connection with the inherent complexity of the polymer repeating unit rather than with the mesoscopic architecture of the whole macromolecule. Indeed, the PNIPAM dynamical transition occurs in both linear chains (19) and microgel networks. (21) Likewise, the transition is observed not only in proteins and nucleic acids but also in amyloid fibrils and even in unbound amino acids. (32,33)
For biological molecules, the possibility of regulating the conformational flexibility across the dynamical transition is of great importance. On one hand, the degree of flexibility achieved thanks to fast picosecond to nanosecond motions is essential for biological agents to reach functionality and the necessary reaction rates at physiological temperatures. (34−38) On the other hand, controlled flexibility and limited biological activity are desirable in the context of protein storage, transport, and delivery because they allow better preservation from denaturation in the case of hostile external conditions, such as high temperatures or low pH. In medicine and pharmaceutics, for instance, careful regulation of the enzyme activity is crucial to improve the shelf life of protein-based drugs. (39−41)
Many ongoing studies aim at elucidating how the atomic motions and conformational flexibility of proteins can be controlled by properly tuning the characteristics of their solvent environment. Water, the main physiological milieu of biomolecules, is well known as the archetype of plasticizers, i.e., the class of solvents that impart the flexibility needed to undergo the dynamical transition and eventually perform biological functions. On the contrary, protein solvation by stabilizing additives, such as sugars and polyols, results in a stiffening of the molecular motions and leads to either a shift of Td toward higher temperatures or complete suppression of the transition. (42−49) Stabilizers are routinely exploited for long-term protein storage, (50) thus reinforcing the connection between molecular flexibility at the picosecond to nanosecond time scale and stability at the macroscopic level. (51,52) Detailed knowledge of how different classes of solvents affect the fast dynamics of macromolecules is therefore of paramount importance. Although the relevant existing literature is wide and keeps growing, the details of the underlying mechanisms are still much debated.
From the perspective of exploiting PNIPAM-based systems as protein analogues, the study of the dynamical transition of PNIPAM in the presence of plasticizing or stabilizing solvents acquires a relevant and 2-fold value. On one hand, it can provide new insights onto the solute–solvent interactions at the basis of the dynamical transition observed in complex macromolecules. On the other hand, this study will help to push further the PNIPAM–protein analogy and possibly open new routes to artificially replicate biological functions by means of tailored smart materials.
In this work, we study how the addition of stabilizing agents, like sugars (glucose) and polyols (glycerol), impacts the dynamical transition of hydrated PNIPAM chains. For this purpose, we performed an extensive campaign of elastic incoherent neutron scattering (EINS) measurements that allowed us to derive the MSDs of PNIPAM in a time interval ranging from 150 to 2000 ps. From the analysis of both EINS intensities and MSDs, a coherent picture emerged about the impact that the solvent characteristics have on both the onset temperature of the dynamical transition and the amplitude of the relevant atomic motions. We found that when the polymer is embedded in glycerol or glucose, the dynamical transition occurs even in the absence of water, although an upshift of the transition temperature Td is observed. In the presence of binary solvent mixtures, the competition between water and cosolvent in interaction with PNIPAM results in a strong dependence of Td on the water content. Water–PNIPAM interactions appear favored down to rather low hydration levels, suggesting a preferential affinity of the polymer for water molecules. Remarkably, the comparison with proteins reveals the existence of strong analogies between PNIPAM and protein behavior, not only in purely hydrated conditions but even in the presence of more complex solvent environments.

2. Materials and Methods

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2.1. Samples

PNIPAM linear chains (number-averaged molecular weight Mn = 40 kDa), glucose-1,2,3,4,5,6,6-d7 (97 atom % D), and glycerol-d8 (≥98 atom % D) were purchased from Sigma-Aldrich. Deuterium oxide (D2O, 99.96 atom % D) was purchased from Cambridge Isotopes Laboratories, Inc. Samples of PNIPAM, PNIPAM in deuterated glycerol (PNIPAM:d-glyc), and PNIPAM in deuterated glucose (PNIPAM:d-glu) were prepared at different values of hydration degree h, defined as the ratio between the mass of heavy water mw and the mass of PNIPAM mP. From here onward, the hydration degree h = 0.35, for instance, will be referred to as 0.35h. The list of prepared samples together with the corresponding hydration degree and other relevant experimental details is reported in Table 1. Each sample was prepared starting with the same amount of PNIPAM mP ≈ 250 mg. An equal amount of cosolvent mc ≈ 250 mg was added where required in order to have a fixed 1:1 weight ratio with PNIPAM, in analogy with the procedure typically used for the preparation of protein–glycerol and protein–glucose mixtures. (44,53) The powder samples were then dissolved in ≈5 g of D2O and cool stored at about 277 K for 2 days to ensure that all exchangeable hydrogen atoms were properly substituted with deuterium. The samples were subsequently freeze dried and then further dehydrated under vacuum in the presence of P2O5 until a stable dry weight was achieved. The dry samples were put in the presence of D2O vapor until the desired value of h was reached. Samples were finally sealed with indium wire in standard aluminum flat cells. For PNIPAM:d-glu 0h and 0.15h, a lead wire was used instead to allow these two samples to be measured up to higher temperatures (see Table 1). The inner thickness t of each cell was individually chosen to obtain a theoretical neutron transmission of about 90% for every sample (see Table 1).
Table 1. List of Samples Together with Specific Details of Sample Preparation and Other Experimental Information
       [K]f
samplehabtc [mm]d [%]eIN13 τR = 150 psSPHERES τR = 2000 ps
PNIPAM 0h0.05 0.290.792153–31320, 153–298
PNIPAM 1h1.00 0.391.684153–29820, 153–298
PNIPAM:d-glyc 0h0.0510.392.784153–31820, 153–318
PNIPAM:d-glyc 0.15h0.1510.392.283153–31820, 153–298
PNIPAM:d-glyc 0.35h0.3510.490.782153–31820, 153–298
PNIPAM:d-glyc 1h1.0010.492.178153–31820, 153–298
PNIPAM:d-glu 0h0.0510.390.685153–40320, 153–418
PNIPAM:d-glu 0.15h0.1510.390.584153–39320, 153–398
PNIPAM:d-glu 0.35h0.3510.392.082153–31820, 153–298
PNIPAM:d-glu 1h1.0010.490.678153–31820, 153–298
a

Measured value of hydration degree. The estimated relative error is of about 5%.

b

Cosolvent/PNIPAM weight ratio.

c

Cell inner thickness.

d

Theoretical neutron transmission.

e

Ratio between incoherent and total scattering cross section.

f

Measured temperature ranges.

2.2. Elastic Incoherent Neutron Scattering Concepts

In neutron scattering experiments, the observable quantity is the dynamic structure factor S(Q, E), which provides the probability for a neutron impinging on the sample to be scattered with a wavevector transfer Q and an exchanged energy E. For isotropic (amorphous) samples, like those considered in this study, the dynamic structure factor only depends on the magnitude Q of the wavevector transfer. The interaction of (thermal) neutrons with matter is such that S(Q, E) contains both coherent scattering contributions, arising from spatial correlations between different atoms as a function of time, and incoherent scattering contributions, due to spatial correlations of a single atom with itself as a function of time and averaged over all atoms. As such, the two contributions provide information about collective and individual atomic movements, respectively.
Since the incoherent scattering cross section of hydrogen (σincH ≈ 80 barn) is much larger than both the coherent and the incoherent cross sections of any other element (a few barns), the incoherent contribution of the hydrogen atoms is largely dominant in our samples (see Table 1). In particular, we exploited the large difference between the hydrogen and the deuterium cross section (σtotD ≈ 8 barn) by using deuterated solvents and cosolvents, to highlight the dynamics of PNIPAM nonexchangeable H atoms with respect to the dynamics of the solvent constituents. As H atoms are uniformly distributed over the polymer repeating unit (see Scheme 1), eventually they bear information about the dynamics of the PNIPAM chains as a whole.
In a real experiment, the measured dynamic structure factor is convoluted with the resolution function of the employed spectrometer. In the energy domain, the resolution function in the proximity of the elastic peak can often be approximated by a Gaussian shape, characterized by a full width at half-maximum (fwhm) ΔE, whose value depends on the chosen spectrometer and setup and which also defines the instrumental resolution time scale . In the case of elastic incoherent neutron scattering (EINS), the convolution with the resolution function implies that the dynamic structure factor is measured and integrated within a narrow energy window |E| ≲ ΔE around the elastic condition E = 0. The resulting EINS intensity is named “elastic” in the sense that only sample atomic motions with characteristic time scales slower than τR─and thus closer in energy to the elastic line─contribute to the measured signal, whereas faster motions fall outside the measured energy window. Any physical or chemical process inducing an increase of atomic mobility, such that the time scales of the involved motions change from larger to smaller values than τR, results in a decrease of .

2.3. Elastic Incoherent Neutron Scattering Experiments

EINS intensities were collected on two neutron backscattering spectrometers with different energy resolutions ΔE, namely, SPHERES, (54,55) operated by JCNS at the Heinz Maier–Leibnitz Zentrum (MLZ) in Garching (Germany), and IN13, (56) at the Institut Laue–Langevin (ILL) in Grenoble (France). SPHERES is characterized by an incident energy Ei = 2.08 meV and a resolution time scale τR of about 2000 ps (ΔE ≈ 0.6 μeV fwhm). Data were collected in the available range of exchanged momentum Q between 0.22 and 1.84 Å –1. Compared to SPHERES, IN13 is characterized by a higher incident energy Ei = 16.45 meV, which allows one to explore a wider Q range between 0.19 and 4.99 Å –1. The resolution time scale of IN13 is τR ≈ 150 ps (ΔE ≈ 8 μeV fwhm).
The temperature ranges in which the samples were measured are reported in Table 1. On IN13, EINS intensities were collected in constant temperature acquisitions with ΔT steps ranging between 5 and 30 K depending on the expected temperature evolution of each sample. On SPHERES, thanks to its higher flux, EINS intensities were collected along temperature ramps with a 0.23 K/min heating rate, resulting in acquisition steps of about 2 K. EINS intensities on SPHERES were also collected at T = 20 K in order to have a low-temperature value for data normalization.
Transmission measurements confirmed the values calculated for the chosen sample thicknesses (as reported in Table 1). Data were corrected for cell scattering, self-shielding, and detector efficiency by vanadium (IN13) or low-temperature (SPHERES) normalization, whereas multiple-scattering events were neglected. Noisy detectors, mostly located at the highest and lowest angles, were removed. The fully corrected data finally span the Q ranges 1.27–3.93 Å–1 for IN13 and 0.60–1.76 Å–1 for SPHERES.

2.4. Fitting Model

From the EINS intensities, the atomic MSDs can be derived by assuming specific functional forms for depending on the type of active motions. At sufficiently low temperatures, large-amplitude macromolecular motions are essentially frozen and only small-amplitude harmonic vibrations can take place, which are properly described by a set of quantized harmonic oscillators. (57) In this case, takes a Gaussian Q dependence (58)
(1)
where S0 is an intensity prefactor accounting for instrumental details. The term A(T) is proportional to the harmonic MSD, which can be assumed to increase roughly linearly with temperature, (59) according to the well-known Debye model. (60) The Gaussian approximation is strictly valid only at low Q values. (61) Moreover, it becomes more and more inadequate on increasing temperature because a variety of nonharmonic motions, such as diffusions, relaxations, side-chain fluctuations, and structural and conformational transitions, become progressively active. The larger number of degrees of freedom allows the macromolecule to assume many equivalent local configurations (or conformational substates (62)) while maintaining the same coarse structure. The wider exploration of conformational space can be described in a simplified way as jumps between two potential wells separated by an effective distance d. Such a double-well model has been successfully used to describe the dynamical transition of biomolecules including proteins, (43,44,53,63) homomeric polypeptides, (64) and DNA (24) as well as that of PNIPAM microgels (21) and linear chains. (19) In this model, takes the form (65)
(2)
where p1 and p2 are the probabilities of finding the atom in the ground and excited wells, respectively.
Using a Gaussian approximation, the 3-dimensional, isotropic MSDs ⟨Δu2⟩tot can be written as (19,45)
(3)
Well below the dynamical transition, ⟨Δu2⟩tot increases approximately linearly with T since the MSDs are dominated by the harmonic term A(T). When the system is heated up, the probability p2 of exploring the excited state increases until the second term in eq 3 becomes dominant, resulting in a steep increase in the slope of ⟨Δu2⟩tot at Td.
All of the parameters in eq 2 may in principle depend on the temperature, sample composition, or instrumental time scale. Preliminary fitting of the data showed a substantial independence of the distance parameter d from the temperature and solvent composition over the whole set of samples, whereas it significantly depended on the instrumental time scale. At τR = 150 ps, a nearly constant value of d ≈ 1.3 Å was found for all samples, close to the values of d reported for lysozyme and DNA at the same time scale. (24,44,47,63) At τR = 2000 ps, a larger value of d ≈ 2.4 Å was found. This is consistent with the basic idea that PNIPAM atoms can span a wider portion of the conformational space over longer time scales. It should be also considered that the intensities collected at τR = 150 and 2000 ps cover a higher and lower Q region, respectively. Correspondingly, the two data sets mainly provide information on shorter and larger length scales, consistent with the obtained values of d (≈1.3 Å at τR = 150 ps, ≈2.4 Å at τR = 2000 ps).
On the basis of these preliminary evaluations, we developed an analysis method to simultaneously fit the whole collection of data, thus significantly improving the final error bars of the resulting MSDs. In setting up such a global fitting procedure, we introduced the following constraints.
(i)

The parameter d was assumed to be independent of temperature and solvent composition. The dependence of d on the resolution time scale τR was instead expressed through a diffusion-like form d = ϕτRξ. (64,66) In this formula, ϕ is related to a diffusion coefficient and ξ takes into account a possible subdiffusive character.

(ii)

The harmonic contribution in eq 3 was assumed to increase linearly with T in the temperature window of interest; therefore, it was expressed as A(T) = aT + C. Moreover, we assumed the harmonic dynamics to be equally resolved by the two instruments, since we expect its characteristic time scale to be much faster than the employed τR, and thus, the parameters a and C were imposed to be independent of τR.

(iii)

Considering the above assumptions, ξ, ϕ, a, and C were constrained to be global fitting parameters, taking the same value for the whole data set. The probability product p1p2 and the prefactor S0 were instead unconstrained and left free to vary as a function of T, τR, and solvent composition.

After several tests, all of the collected intensities were simultaneously fitted over a fixed Q range, namely, between 1.27 and 3.93 Å–1 at τR = 150 ps and between 0.60 and 1.42 Å–1 at τR = 2000 ps. Examples of the resulting fitting functions are reported in Figure 1 for PNIPAM:d-glyc 1h, together with the corresponding experimental EINS intensities. For the sake of comparison, also the purely Gaussian fit is reported. The comparison shows how the double-well model of eq 2 captures much better the behavior of the EINS intensities over the wide explored range of Q, T, and h, especially at high temperatures.

Figure 1

Figure 1. Logarithm of the EINS intensities of PNIPAM:d-glyc 1h as a function of Q2 at selected temperatures. Experimental data are reported together with the double-well fitting functions (solid red lines). For comparison, corresponding Gaussian fitting functions (dashed green lines) are also reported.

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The resulting fitting parameters were used to determine the final MSDs by means of eq 3. The MDSs reproduced the behavior qualitatively obtained from the preliminary fit but with clearer and more robust trends. Further details on the data fitting procedure are reported in the Supporting Information.

3. Results

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3.1. Elastic Incoherent Neutron Scattering Intensities

Preliminary information on the dynamics of PNIPAM is already provided by the inspection of the Q-integrated EINS intensities as a function of temperature, , reported in Figure 2 for the whole set of samples. For a better comparison between data collected at different τR values, the intensities were obtained by integrating the corresponding over the restricted Q range 1.27–1.76 Å–1, which is common to the two instruments.

Figure 2

Figure 2. Logarithm of the integrated EINS intensities versus T for (a) PNIPAM, (b) PNIPAM:d-glyc, and (c) PNIPAM:d-glu. Data are rescaled by their value at T = 153 K. Gray bars and black straight lines are guides for the eye, helping to visualize the slope changes at Td occurring in the 1h sample of each series. Inset in (a): comparison between the of PNIPAM 1h collected on two different spectrometers.

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≈
Inspection of allows a first identification of Td by detection of the slope changes witnessing the activation of the sample dynamics. Although providing only a first qualitative indication, this evaluation has the advantage of being model independent. On comparing the behavior of PNIPAM, PNIPAM:d-glyc, and PNIPAM:d-glu at the highest hydration degree (1h), a slope change can be observed at Td in the temperature range between 220 and 240 K for all three systems, independently from the presence of cosolvents. Conversely, in the anhydrous samples (0h), the dynamical transition is strongly affected by the solvent composition: it is substantially suppressed in pure PNIPAM, whereas it occurs at ≈270 K in PNIPAM:d-glyc and at ≈370 K in PNIPAM:d-glu. In the other samples with cosolvents, at the intermediate hydration degrees between 0h and 1h, increasing the water content Td progressively decreases from its value in the anhydrous sample to ≈230 K.
Further inspection of reveals also the presence of additional slope changes besides Td. In pure PNIPAM at 1h (panel a), a drastic intensity decrease is observed at ≈270 K. The sharpness of the drop suggests the occurrence of a structural phase transition such as melting of D2O rather than a dynamical effect. Indeed, calorimetric studies on hydrated PNIPAM (67,68) and, in particular, recent investigations on D2O-hydrated PNIPAM chains with the same molecular weight of those considered herein (69) showed that, in samples with h = 1, a fraction of water molecules undergoes cold crystallization on heating and subsequently melts at ≈270 K. A smaller fraction of molecules undergoes crystallization upon cooling and melts at ≈277 K on heating with the total amount of crystalline water however being quite low. (69) In the context of hydrated polymers, cold crystallization is associated with “weakly-bound” water, i.e., a population of water molecules forming with polymer chains an interaction of intermediate strength between that of free bulk molecules, which crystallize upon cooling, and bound hydration molecules, which never crystallize. (70−72) Upon further heating such cold-crystallized molecules eventually melt, but the melting temperature is lower than that in the bulk because of the confining effect due to the interaction with polymer chains. (73,74) The intensity drop around 270 K in our PNIPAM 1h sample can therefore be ascribed to the coupling of the polymer dynamics with melting of weakly bound water molecules. Above ≈275 K, when the phase transition is completed, the intensity slope goes back to a nearly constant linear trend. A similar effect is also observed in PNIPAM:d-glyc 1h (Figure 2b) and PNIPAM:d-glu 1h (Figure 2c), respectively, around 260 and 270 K, suggesting the occurrence of a structural phase transition also in these two samples.
Moving back to the low-temperature region, the data of pure PNIPAM (Figure 2a) show that for the dry sample is slightly but systematically lower than that for the hydrated one, indicating a higher rigidity of the hydrated polymer. Similar evidence is also found in proteins, (38,61,75) DNA, (76) and small peptides, (77) for which it has been suggested that at low temperatures frozen water reduces the MSDs of the macromolecule by constraining its fast fluctuations.
A last interesting observation arises from the comparison between values of at different τR. As reported in the inset of Figure 2a, in PNIPAM 1h the overall intensity decrease as a function of T is more pronounced for longer τR values, consistent with the fact that a larger fraction of moving atoms gets out of a smaller elastic window.
The qualitative picture emerging so far suggests a tight coupling between the dynamics of PNIPAM and that of the surrounding solvent. More quantitative information can be obtained from the analysis of the Q dependence of the EINS intensities, as detailed in the following.

3.2. Mean Square Displacements

The MSDs of each sample, ⟨Δu2⟩tot, were obtained by fitting the Q dependence of the corresponding set of EINS intensities using the procedure reported in the Materials and Methods. As described in this section, the double-well distance d was constrained to follow the behavior d = ϕτRξ. The analysis provides the parameter values ξ = 0.251 ± 0.003 and ϕ = (0.361 ± 0.008) Å/psξ (see Table 2), with ξ < 0.5 indicating a subdiffusive motion. (64,66) The corresponding values of d are 1.27 ± 0.03 Å at τR = 150 ps and 2.43 ± 0.06 Å at τR = 2000 ps. For comparison, several studies showed that the dynamical transition in proteins involves the activation of localized motions between ≈1.5 and ≈3 Å, (75,78) associated with large-amplitude backbone and side-chain fluctuations. (79,80) The role of backbone and side-chain dynamics in the dynamical transition has been highlighted also in PNIPAM microgels. (20)
Table 2. Values of d, ϕ, ξ, a, and C Obtained from the Fit on the Whole Data Set with the Corresponding Reduced χ2
τR [ps]ξϕ [Å/psξ]d [Å]a [Å2 × 10–4/K]C [Å2]χ2
1500.251 ± 0.0030.361 ± 0.0081.27 ± 0.031.95 ± 0.040.006 ± 0.0023.2
2000  2.43 ± 0.06   
The dynamical transition temperature Td of each sample was defined as the intersection between two straight lines obtained by fitting ⟨Δu2⟩tot in two temperature ranges, respectively, below and above the corresponding slope change. The resulting values of Td are reported in Table 3. In the following, we will separately analyze the MSDs of PNIPAM in each of the three investigated environments.
Table 3. Dynamical Transition Temperatures Td As Obtained from the Linear Fit of the MSDs
 Td [K]
sampleτR = 150 psτR = 2000 ps
PNIPAM 0h  
PNIPAM 1h220 ± 2220 ± 1
PNIPAM:d-glyc 0h270 ± 5270 ± 1
PNIPAM:d-glyc 0.15h248 ± 6248 ± 1
PNIPAM:d-glyc 0.35h225 ± 5230 ± 2
PNIPAM:d-glyc 1h218 ± 4219 ± 2
PNIPAM:d-glu 0h357 ± 2380 ± 5
PNIPAM:d-glu 0.15h298 ± 6301 ± 2
PNIPAM:d-glu 0.35h255 ± 5 
PNIPAM:d-glu 1h225 ± 11234 ± 1

3.2.1. Pure PNIPAM

The MSDs of pure PNIPAM are reported in Figure 3. In agreement with the larger value assumed by d at longer τR, the resulting ⟨Δu2⟩tot are greater at τR = 2000 ps than at τR = 150 ps.

Figure 3

Figure 3. ⟨Δu2⟩tot of pure PNIPAM. For easier comparison of their slope, data were corrected for their value at 153 K (unsubtracted data are shown in the insets). Red lines result from the linear fit of ⟨Δu2⟩tot for the 1h sample. Arrows indicate the slope change at Td.

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The MSDs of PNIPAM 0h do not show abrupt changes in the slope, consistent with the fact that the dynamical transition is essentially suppressed when the system approaches dry conditions, as widely reported for various biomolecules (80,81) and, more recently, for PNIPAM microgels (21) and linear chains. (19) At low temperatures, a slight deviation of the MSDs from a straight linear trend can be observed especially at τR = 2000 ps, as they smoothly inflect over a broad temperature range between ≈153 and ≈200 K. The behavior of the MSDs might be reminiscent of the activation of the rotational dynamics of methyl groups, typical of PNIPAM-based systems (20) as well as of other polymers and biological molecules. (25,47,80−82) Methyl dynamics becomes active between 100 and 150 K on the time scales accessible to neutron scattering, independent from the presence and composition of the solvent. Indeed, methyl rotations are characterized by a rather wide distribution of activation energies, yielding a distribution of activation temperatures which eventually results in a smeared MSDs response. (81,82) Considering this, we can infer that although the start of the methyl rotation processes occurs at temperatures lower than those investigated in this paper, the ending tail of methyl dynamics activation can still be observed in our temperature range.
In PNIPAM 1h, a dynamical transition clearly takes place at Td ≈ 220 K (see Table 3), similarly to hydrated biomolecules. (24,26,80,81) A close value of about 225 K has been previously reported for PNIPAM chains at 0.67h, (19) suggesting that Td remains substantially unaffected when the hydration level increases from 0.67h to 1h. This allows one to separate the dynamical transition from the glass transition of hydrated PNIPAM, reported by calorimetric investigations. (67,69) Contrary to Td, the glass transition temperature Tg of PNIPAM shows a strong dependence on h, and in particular, it shifts from 243 K at 0.67h to 230 K at 1h. A distinction between glass and dynamical transitions has also been pointed out for hydrated proteins, where the former is reflected in a mild slope change in the MSDs at Tg ≈ 200 K followed by a more marked change at Td due to the dynamical transition. (28) However, the observation of the slope change at Tg has proven elusive since in most cases it is hidden by the occurrence of the sharper dynamical transition.
At low temperatures, the MSDs of PNIPAM 1h are smaller than those in the dry sample. As discussed for the corresponding EINS intensities, the decreased mobility of hydrated PNIPAM is probably caused by the constraining action exerted on the polymer by frozen water: since the layer of frozen water surrounding the polymer is stiffer than the polymer itself, also the water–PNIPAM mixture is on average stiffer and thus “more harmonic” than the polymer alone. In the same sample, the drastic jump of the MSDs at ≈270 K (see insets of Figure 3) is related to the fact that, on heating, some hydration water molecules cold crystallize and subsequently melt within a narrow temperature range around 270 K with consequent release of PNIPAM degrees of freedom.

3.2.2. PNIPAM with Glycerol

The MSDs of PNIPAM:d-glyc at τR = 150 and 2000 ps are shown in Figure 4a and 4b, respectively. Contrary to the case of pure PNIPAM, a dynamical transition occurs even in the absence of water (0h) at Td ≈ 270 K (see Table 3). On adding water, Td progressively shifts toward lower values, passing from ≈250 K at 0.15h to ≈230 K at 0.35h and ≈220 K at 1h.

Figure 4

Figure 4. ⟨Δu2⟩tot of PNIPAM:d-glyc at τR = 150 (a) and 2000 ps (b). MSDs are corrected for their value at T = 153 K. Solid lines and colored bars help to visualize for each sample the slope changes at Td. Further slope changes at Tin are indicated by arrows. In the inset, slope changes at Tg are highlighted.

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Below Td, for τR = 150 ps, a further and slight change in slope is observed in the 0h and 0.15h samples at Tg = 206 ± 7 and 196 ± 10 K, respectively (see inset of Figure 4a). Identification of this slope change with the dynamical transition is unlikely because the presence of glycerol is expected to slow down the dynamics of PNIPAM, and therefore, Td should not occur below the value of ≈220 K found in pure water. Considering that the glass transition of the corresponding water/glycerol mixtures falls around 190 and 180 K, respectively, (83) the observed change may be related instead to a coupling with the solvent glass transition. This interpretation has been proposed to explain a similar slight change at ≈200 K in the MSDs of lysozyme in pure glycerol. (28)
Above Td, closer inspection of the MSDs of PNIPAM:d-glyc 1h reveals an additional slope increase at Tin ≈ 230 K followed by a slope decrease at Tm ≈ 250 K. To assess whether this behavior is related to a solvent phase transition as in the case of PNIPAM 1h, we performed calorimetric measurements on this sample (see Figure S5 of the Supporting Information). The thermogram shows a broad endothermic peak in the same temperature range, which is compatible with a progressive solvent melting process, starting at about Tin and ending at Tm. The observation of a large peak is consistent with the fact that water/glycerol mixtures alone are rather polymorphic systems, where multiple and partially overlapping solid-state phase changes are observed. (84) Moreover, the interaction with PNIPAM can induce local fluctuations of the nominal solvent composition with a consequent distribution of melting temperatures, which can further explain the observed broadening of the melting peak. The MSDs behavior between Tin and Tm is well consistent with the calorimetric data and the progressive melting of such distributions of solid phases and/or solvent compositions.

3.2.3. PNIPAM with Glucose

The MSDs of PNIPAM:d-glu are shown in Figure 5. Compared to PNIPAM and PNIPAM:d-glyc, the slightly nonlinear trend at low temperatures that we attributed to methyl dynamics is now more pronounced, as especially evident at τR = 2000 ps (Figure 5b).

Figure 5

Figure 5. ⟨Δu2⟩tot of PNIPAM:d-glu at τR = 150 (a) and 2000 ps (b). MSDs are corrected for their value at T = 153 K. Solid lines and colored bars help to visualize for each sample the slope changes at Td. Further slope changes at Tin are indicated by arrows. In the insets, the slope changes at Tg are highlighted.

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A dynamical transition is observed at all of the investigated h values, and the corresponding Td values are reported in Table 3. In the anhydrous sample (0h), the transition occurs at very high temperatures for both time resolutions, namely, ≈360 K for τR = 150 ps and ≈380 K for τR = 2000 ps. In the latter case, a further and sudden increase of the MSDs appears above 400 K, close to the melting temperature of pure glucose. This suggests the formation of crystalline domains within an otherwise amorphous sample. Indeed, cold crystallization of amorphous glucose is observed roughly between 360 and 380 K in the presence of trace amounts of water followed by melting. (85) In this sample, cold crystallization of glucose might also explain the upshift of Td with respect to PNIPAM:d-glu 0h measured at τR = 150 ps since the increased rigidity due to cold crystallization would compete with the increased mobility due to the dynamical transition, thus retarding to 380 K the observation of Td.
On increasing h, Td shifts to ≈300 K at 0.15h, ≈260 K at 0.35h, and ≈230 K at 1h. The PNIPAM:d-glu 0.35h sample measured at τR = 2000 ps constitutes a peculiar case because the MSDs do not show any marked change in slope that can be associated with the dynamical transition. In fact, at this hydration level, Td gets close to the concomitant low-temperature inflection associated with methyl groups dynamics, which can thus prevent clear observation of the dynamical transition. In PNIPAM:d-glu 1h, the onset of the transition becomes visible again as a slope change despite the methyl contribution since the higher water content enhances the MSDs increase due to the activation of conformational dynamics.
Below Td, a mild slope change is observed at τR = 150 ps in the 0h, 0.15h, and 0.35h samples, respectively, at Tg = 309 ± 7, 261 ± 6, and 202 ± 11 K (see the inset of Figure 5a). A slope change at Tg = 260 ± 7 K is observed in PNIPAM:d-glu 0.15h also at τR = 2000 ps (inset of Figure 5b). In analogy with the case of PNIPAM:d-glyc, this might be due to a coupling of the fast dynamics of PNIPAM with the glass transition of the solvent. By comparison, the glass transition temperature of the corresponding solvent mixture falls at about 300, 240, and 200 K, respectively. (86) A similar slope change at Tg ≈ 300 K is observed also in the MSDs of lysozyme in pure glucose. (28)
In the MSDs of the 1h sample, a further slope increase above Td is observed at Tin ≈ 250 K followed by an abrupt slope decrease at Tm ≈ 260 K. Similarly to the case of PNIPAM:d-glyc 1h, we interpret Tin and Tm, respectively, as the starting and ending points of a solvent melting process, as confirmed by calorimetric data (see Figure S6 of the Supporting Information).

4. Discussion

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Overall, the above findings reveal a complex behavior of the PNIPAM atomic dynamics as a function of solvent composition, reflecting the complex interplay of microscopic interactions between PNIPAM and water, PNIPAM and cosolvent, and water and cosolvent. The most striking result is that in the presence of a cosolvent Td strongly depends on the hydration level h: as shown in Figure 6, on increasing water content, Td tends to its value without cosolvents and reaches it already at 1h. In light of this, it is convenient to discuss our results by separating those obtained in the high-hydration regime (h ≈ 1) from those in the low-hydration regime (h ≲ 0.4). In the high-hydration regime, water-mediated interactions are predominant, as suggested by the fact that Td behaves as if the cosolvent were not present. In the low-hydration regime instead, where Td strongly depends on the solvent composition, we expect PNIPAM–cosolvent interactions to be more effective.

Figure 6

Figure 6. Dynamical transition temperatures Td of (a) PNIPAM:d-glyc and (b) PNIPAM:d-glu as a function of h. Dashed red lines indicate the value of Td in PNIPAM 1h (without cosolvents).

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4.1. High-Hydration Regime

In PNIPAM–water–cosolvent samples at 1h, the value of Td is close to that found in the PNIPAM–water sample without cosolvent (Figure 6). This suggests that in ternary samples when the hydration level is sufficiently high, cosolvent molecules tend to be preferentially excluded from the PNIPAM solvation shell with the polymer being therefore preferentially hydrated. Such a mechanism of preferential hydration has already been proposed for the interaction between sugars and PNIPAM in diluted solutions. (12,87) Recent investigations, in particular, showed how the addition of a sugar (trehalose) to aqueous PNIPAM results in a preferential exclusion of the cosolvent from the polymer surface, with the sugar developing strong interactions with water molecules while the polymer hydration state is preserved. (12) Our results extend the preferential exclusion mechanism to much more concentrated PNIPAM dispersions as well as to the case of a polyol cosolvent like glycerol.
Preferential exclusion of stabilizing compounds is observed also in aqueous solutions of proteins. (88−90) In particular, an h dependence of Td similar to that shown in Figure 6 has been observed for lysozyme embedded in water/glycerol and water/glucose matrices, where Td reaches its limiting value for h ≳ 0.4. (43−45) On comparing the two systems, it seems that preferential exclusion starts to take place at similar values of h in both PNIPAM and lysozyme, which suggests that PNIPAM linear chains have a capability to coordinate and confine water molecules comparable to proteins. This is likely an essential requisite for the “biomimic” character of PNIPAM, especially considering that it is precisely this kind of confined, proximal water that plays a major role in determining protein functional dynamics. (23)
From the MSDs, an estimate of the polymer rigidity can be obtained by means of the pseudoforce constant (91), where k is expressed in N/m if ⟨Δu2⟩tot is given in Å2 and T in K. In general, we expect k to depend on both the solvent composition and the temperature since it will be influenced by the activation of anharmonic processes and by phase transitions. By looking at the MSDs reported in Figure 7, three different temperature regions to calculate k can be identified in each sample: (1) a low-temperature region, below the dynamical transition temperature Td; (2) an intermediate-temperature region, above Td and below the start of solvent melting at Tin; (3) a high-temperature region above Tm, where the melting transition is completed.

Figure 7

Figure 7. MSDs of PNIPAM, PNIPAM:d-glyc, and PNIPAM:d-glu in the high-hydration regime (1h). MSDs are corrected for the corresponding value at T = 153 K. For each sample, the corresponding values of Td, Tin, and Tm are indicated by arrows. Solid lines are obtained from the linear fit of the data in the temperature region below Td (1), above Td and below Tin (2), and above Tm (3).

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In the low-temperature region, where only harmonic and methyl dynamics are active, the MSDs are small, indicating low flexibility, and the associated pseudoforce constants are rather large, as shown in Figure 8. A larger k is observed in the presence of cosolvents, especially in the case of glucose and for τR = 2000 ps, where the data have better statistics and a finer temperature sampling.

Figure 8

Figure 8. Pseudoforce constant k at τR = (a) 150 and (b) 2000 ps calculated for PNIPAM samples at 1h. Values of k are obtained from the MSDs slope in the temperature region (1) below Td (red), (2) between Td and Tin (blue), and (3) above Tm (yellow).

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In the intermediate-temperature region, the occurrence of the dynamical transition leads to an increase of the MSDs slope in all samples, and a corresponding decrease of k is observed for both of the investigated τR values. As in the previous case, higher values of k are found in the presence of cosolvents and the effect is larger for glucose. The stronger constraining action of glucose on the dynamics of PNIPAM is also witnessed by the fact that it shifts Td to a value about 10 K higher than in the other two hydrated samples.
In the high-temperature region, the presence of cosolvents is particularly influential in determining the value of k. On one hand, in the purely hydrated sample, the pseudoforce constant decreases from the values assumed in the low- and intermediate-temperature regions at both τR values. On the other hand, in the presence of cosolvents, k increases, indicating that the polymer becomes more rigid than in the intermediate-temperature regime, as especially evident at τR = 2000 ps. Such a reduced mobility of PNIPAM at high temperature might be explained by a temperature effect on the preferential hydration mechanism. Indeed, preferential hydration in these samples should lead to the presence of a water-rich environment around the polymer, which, below melting, might allow PNIPAM to be relatively flexible. Above melting, solvent molecules diffuse more freely; therefore, preferential hydration might become less effective and PNIPAM might sense a more homogeneous and viscous environment. A further contribution to the increased polymer rigidity at high temperature can derive from the transition of PNIPAM to the more compact globular conformation above the LCST, as mentioned in the Supporting Information. It is worth noting that recent computational investigations on the PNIPAM–water–trehalose system at T = 283 and 318 K showed how preferential exclusion of trehalose from the PNIPAM solvation shell is less effective at the highest investigated temperature (when the polymer is in the globule conformation). (12)

4.2. Low-Hydration Regime

We will now examine the behavior of the MSDs of PNIPAM samples in the anhydrous state (0h) reported in Figure 9. At low temperatures, the MSDs of pure PNIPAM are larger than those of PNIPAM embedded in glycerol and glucose. Correspondingly, the pseudoforce constants k are smaller in pure PNIPAM than those in glycerol or glucose (Figure 10). As a whole, both the MSDs and the k values indicate a higher mobility of the pure polymer with respect to PNIPAM embedded in glycerol or glucose. We already observed an analogous behavior for PNIPAM in water alone at 1h, where the MSDs below Td are smaller than those in the dry sample (see Figure 3), and we ascribed this effect to the constraining action exerted by frozen water. We can now conclude that below Td the addition of any of the three investigated solvents results in a stiffening of the polymer, irrespective of the specific nature of the solvent. However, the constraining action of the two cosolvents, and especially of glycerol, in particular, appears more effective than that of water, since the pseudoforce constant k is smaller in PNIPAM 1h than in PNIPAM:d-glyc 0h and PNIPAM:d-glu 0h (see Figure 10). A similar low-temperature antiplasticizing effect of glycerol is observed in proteins, where the increase of rigidity is ascribed to a greater ability of this cosolvent to stabilize the surface moieties compared to water. (92) Antiplasticization of fast motions at low temperatures is especially relevant in the protein context, since it is thought to inhibit protein degradation and thus favor stabilization. (93)

Figure 9

Figure 9. MSDs of PNIPAM, PNIPAM:d-glyc, and PNIPAM:d-glu at the same hydration degree (0h). For each binary sample, the corresponding values of Td and Tg are indicated by arrows. Solid lines are obtained from the linear fit of the MSDs in the temperature range between 153 and 313 K, 153 and 203 K, 153 and 303 K, respectively, for PNIPAM, PNIPAM:d-glyc, and PNIPAM:d-glu at τR = 150 ps. All MSDs were fitted in the same temperature range between 153 and 209 K at τR = 2000 ps.

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Figure 10

Figure 10. Low-temperature pseudoforce constant k at τR = 150 (empty circles) and 2000 ps (solid squares) calculated for dry PNIPAM (0h) compared with values calculated for PNIPAM embedded in pure water (PNIPAM 1h), pure glycerol (PNIPAM:d-glyc 0h), and pure glucose (PNIPAM:d-glu 0h).

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A crossover from an antiplasticizing to a plasticizing effect of glycerol is observed on increasing the temperature well above Td ≈ 270 K with the MSDs of pure PNIPAM becoming progressively smaller than those of PNIPAM:d-glyc. Such a crossover has also been observed in the MSDs of lysozyme embedded in glycerol. (48,94) An extrapolation of the data reported in Figure 9 suggests that a similar crossover might also occur in PNIPAM:d-glu above the corresponding Td ≈ 370 K, although data for pure PNIPAM at such high temperatures were not collected.
In PNIPAM:d-glyc and PNIPAM:d-glu, the absence of water and the consequent shift of Td toward higher values allow the observation of an additional small change of slope at Tg < Td. As mentioned above, such a low-temperature change in proteins is ascribed to a coupling of the biomolecule dynamics with the slow α relaxation and hence the glass transition of the surrounding solvent. (28) A similar slope change should be observed also in hydrated samples. In the latter case, however, the proximity of Tg to Td often does not allow one to clearly separate the two processes.
The dynamical transition instead, which occurs at higher temperatures, is not expected to be coupled with the slow α relaxation of the solvent. On the other hand, for a given solvent, the dynamical transition occurs around the same temperature for PNIPAM, proteins, and DNA: (25,28,45,48,95) for all of these macromolecules, Td falls around 220 K in water, 270 K in glycerol, and 370 K in glucose. This observation strongly supports the idea that also the dynamical transition is a solvent-driven process. Indeed, the most recent interpretations ascribe the dynamical transition to the fast β relaxation of the solvent, whose characteristic time τβ matches the typical instrumental resolutions of neutron backscattering spectrometers. (28,29) In this regard, it should be noted that since τβ decreases with increasing temperature, an upshift of Td should be expected on decreasing τR. (28) However, no appreciable dependence of Td on τR can be unambiguously detected in our set of data. The observation of such an effect might actually be prevented by specific experimental issues, such as the sparse temperature sampling at τR = 150 ps, as well as the superposition of several dynamical processes over the same temperature window. Future investigations are therefore needed to deepen this interesting aspect by means of a wider and more systematic sampling of the dynamical transition in PNIPAM as a function of the instrumental resolution.

5. Conclusions

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Similarly to what was observed in biological polymers, the dynamical transition occurring in PNIPAM is critically affected by the solvent environment. The stabilizing character of glycerol and glucose is reflected by the fact that their addition to nonhydrated PNIPAM causes a shift of Td to higher values compared to the purely hydrated sample. This implies that the low-temperature constraining (antiplasticizing) action exerted by these compounds extends over a wider temperature range with respect to water.
When water is added, on increasing hydration level, the behavior of the system progressively shifts toward that of hydrated PNIPAM without cosolvents. This might indicate that a preferential exclusion of the cosolvent takes place when the system reaches a proper hydration level. This evidence confirms the hypothesis that the mechanism of interaction between PNIPAM and stabilizers in an aqueous environment is mainly water mediated.
Most of the distinctive features of the dynamical transition of biomolecules, including the transition temperature values, are replicated in PNIPAM. Overall, the existence of such strong analogies leads to two remarkable considerations. On one hand, they underline the prominent role of the solvent in the dynamical transition, which exhibits some universal features irrespective of the specific characteristics of the solvated macromolecule. On the other hand, they bear witness to the existence of fundamental mechanisms of solvent–solute interaction that are common to PNIPAM and biomacromolecules. From an essential point of view, the rich phenomenology of proteins in solution can be traced back to the subtle balance between the interactions of the protein polar and nonpolar groups, both among themselves and with solvent molecules, which ultimately leads to the complex biological behavior and functionality of proteins. Therefore, it may be argued that the ability of PNIPAM to replicate protein behavior is intimately connected to the intrinsic complexity of the polymer repeating unit, enabling it to reproduce the rich energy landscape of proteins with a multitude of different conformational substates. Future works may benefit from such unique properties of PNIPAM-based systems with the aim to extend their applications as artificial analogues of natural biomolecules.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.macromol.2c00177.

  • Further details about the fitting strategy; differential scanning calorimetry analysis; comparison between neutron scattering and calorimetric data (PDF)

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Author Information

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  • Corresponding Author
    • Andrea Orecchini - Dipartimento di Fisica e Geologia, Università di Perugia, Via Alessandro Pascoli, 06123 Perugia, Italy;  Dipartimento di Fisica e Geologia, CNR-IOM c/o Università di Perugia, via Alessandro Pascoli, 06123 Perugia, Italy;  Email: [email protected]
  • Authors
    • Benedetta P. Rosi - Dipartimento di Fisica e Geologia, Università di Perugia, Via Alessandro Pascoli, 06123 Perugia, Italy;  Orcidhttps://orcid.org/0000-0001-9745-5826
    • Arianna D’Angelo - Laboratoire de Physique des Solides, CNRS, Université Paris-Saclay, 510 Rue André Rivière, 91405 Orsay, France;  Institut Laue-Langevin, 71 Avenue des Martyrs, 38042 Grenoble, Cedex 9, France
    • Elena Buratti - Dipartimento di Fisica, CNR-ISC c/o Università di Roma La Sapienza, Piazzale Aldo Moro 5, 00185 Roma, Italy;  Orcidhttps://orcid.org/0000-0003-3982-5141
    • Marco Zanatta - Dipartimento di Fisica, Università di Trento, via Sommarive 14, 38123 Trento, Italy
    • Letizia Tavagnacco - Dipartimento di Fisica, CNR-ISC c/o Università di Roma La Sapienza, Piazzale Aldo Moro 5, 00185 Roma, Italy;  Orcidhttps://orcid.org/0000-0002-3492-7766
    • Francesca Natali - Institut Laue-Langevin, 71 Avenue des Martyrs, 38042 Grenoble, Cedex 9, France;  CNR-IOM, OGG, 71 Avenue des Martyrs, 38043 Grenoble, Cedex 9, France
    • Michaela Zamponi - Jülich Centre for Neutron Science at Heinz Maier-Leibnitz Zentrum, Forschungszentrum Jülich GmbH, Lichtenbergstrasse 1, 85747 Garching, Germany
    • Daria Noferini - Jülich Centre for Neutron Science at Heinz Maier-Leibnitz Zentrum, Forschungszentrum Jülich GmbH, Lichtenbergstrasse 1, 85747 Garching, Germany;  European Spallation Source ERIC, Box 176, 221 00 Lund, Sweden
    • Silvia Corezzi - Dipartimento di Fisica e Geologia, Università di Perugia, Via Alessandro Pascoli, 06123 Perugia, Italy
    • Emanuela Zaccarelli - Dipartimento di Fisica, CNR-ISC c/o Università di Roma La Sapienza, Piazzale Aldo Moro 5, 00185 Roma, Italy
    • Lucia Comez - Dipartimento di Fisica e Geologia, CNR-IOM c/o Università di Perugia, via Alessandro Pascoli, 06123 Perugia, Italy;  Orcidhttps://orcid.org/0000-0001-5160-6844
    • Francesco Sacchetti - Dipartimento di Fisica e Geologia, Università di Perugia, Via Alessandro Pascoli, 06123 Perugia, Italy
    • Alessandro Paciaroni - Dipartimento di Fisica e Geologia, Università di Perugia, Via Alessandro Pascoli, 06123 Perugia, Italy
    • Caterina Petrillo - Dipartimento di Fisica e Geologia, Università di Perugia, Via Alessandro Pascoli, 06123 Perugia, Italy
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This publication was supported by the CarESS project, D.R. no. 597, from the University of Perugia. The authors acknowledge the Institut Laue-Langevin (ILL) and the Jülich Centre for Neutron Science (JCNS) at the Heinz Maier-Leibnitz Zentrum (MLZ) for beamtime allocation.

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    Sun, T.; Qing, G. Biomimetic Smart Interface Materials for Biological Applications. Adv. Mater. 2011, 23, H57– H77,  DOI: 10.1002/adma.201004326
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    Biomimetic Smart Interface Materials for Biological Applications
    Sun, Tao-Lei; Qing, Guang-Yan
    Advanced Materials (Weinheim, Germany) (2011), 23 (12), H57-H77CODEN: ADVMEW; ISSN:0935-9648. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Controlling the surface chem. and phys. properties of materials and modulating the interfacial behaviors of biol. entities, e.g., cells and biomols., are central tasks in the study of biomaterials. In this context, smart polymer interface materials have recently attracted much interest in biorelated applications and have broad prospects due to the excellent controllability of their surface properties by external stimuli. Among such materials, poly(N-isopropylacrylamide) and its copolymer films are esp. attractive due to their reversible hydrogen-bonding-mediated reversible phase transition, which mimics natural biol. processes. This platform is promising for tuning surface properties or to introduce novel biofunctionalities via copolymn. with various functional units and/or combination with other materials. Important progress in this field in recent years is highlighted.
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    Inoue, M.; Hayashi, T.; Hikiri, S.; Ikeguchi, M.; Kinoshita, M. Comparison based on statistical thermodynamics between globule-to-coil transition of poly(N-isopropylacrylamide) and cold denaturation of a protein. J. Mol. Liq. 2020, 317, 114129,  DOI: 10.1016/j.molliq.2020.114129
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    Comparison based on statistical thermodynamics between globule-to-coil transition of poly(N-isopropylacrylamide) and cold denaturation of a protein
    Inoue, Masao; Hayashi, Tomohiko; Hikiri, Simon; Ikeguchi, Mitsunori; Kinoshita, Masahiro
    Journal of Molecular Liquids (2020), 317 (), 114129CODEN: JMLIDT; ISSN:0167-7322. (Elsevier B.V.)
    When the temp. T becomes sufficiently low, poly(N-isopropylacrylamide) (PNIPAM) and a protein, resp., cause the globule-to-coil transition and the cold denaturation (i.e., transitions to states comprising more extended structures). It is exptl. known for PNIPAM that the coil state is sol. in water but the globule state is insol. By contrast, both of the cold-denatured and native states of a protein are sol. Using our recently developed statistical-mech. theory combined with mol. models for water, we show that the two structural transitions share phys. the same mechanism but still the difference between PNIPAM and a protein in terms of the solubilities of the two states can be reproduced. The solute hydration can be decompd. into the two processes: the creation of a cavity matching the solute structure at the at. level in water (process 1: hydrophobic hydration); and the incorporation of solute-water van der Waals interaction potential followed by that of solute-water electrostatic interaction potential (process 2). The hydration free energies, energies, and entropies in processes 1 and 2 are denoted by μH,1 > 0 and μH,2 < 0, εVH,1 < 0 and εVH,2 < 0, and SVH,1 < 0 and SVH,2 < 0, resp. We find that the excluded-vol. (EV) terms in εVH,1 and SVH,1 are strongly dependent on T, whereas not only the sum of the water-accessible surface terms in εVH,1 and SVH,1 but also εVH,2 and SVH,2 remain essentially const. against a change in T. The EV term of SVH,1 becomes significantly smaller at low T, which is interpretable as the weakening of the hydrophobic effect and the trigger of the two structural transitions. The changes in structure and properties of water near PNIPAM or a protein upon the transition to a state comprising more extended structures are unimportant. Though μH,1 is a largely increasing function of T, |μH,2| is only very weakly dependent on T. μH,1/|μH,2| for PNIPAM is much larger than that for a protein, which is attributable to the lower electrostatic affinity of PNIPAM for water. As a consequence, μH(Coil) < 0 at low T but μH(Globule) » 0 at high T for PNIPAM but μH(Denatured) « 0 at low T and μH(Native) « 0 at high T for a protein (μH = μH,1 + μH,2).
  8. 8
    Inoue, M.; Hayashi, T.; Hikiri, S.; Ikeguchi, M.; Kinoshita, M. Mechanism of globule-to-coil transition of poly(N-isopropylacrylamide) in water: Relevance to cold denaturation of a protein. J. Mol. Liq. 2019, 292, 111374,  DOI: 10.1016/j.molliq.2019.111374
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    Mechanism of globule-to-coil transition of poly(N-isopropylacrylamide) in water: Relevance to cold denaturation of a protein
    Inoue, Masao; Hayashi, Tomohiko; Hikiri, Simon; Ikeguchi, Mitsunori; Kinoshita, Masahiro
    Journal of Molecular Liquids (2019), 292 (), 111374pp.CODEN: JMLIDT; ISSN:0167-7322. (Elsevier B.V.)
    In water, poly(N-isopropylacrylamide) (PNIPAM) is in a sol. coil state below the lower crit. sol. temp. (LCST) but in an insol. globule state above LCST. Namely, as the temp. decreases, PNIPAM exhibits a globule-to-coil transition at LCST∼305 K. We generate structural ensembles of coil and globule states by all-atom mol. dynamics simulations conducted at 273 and 323 K, resp. We then calc. a variety of energetic and entropic components of thermodn. quantities of the two states at the two temps. using our recently developed, accurate statistical-mech. method for solute hydration where mol. models are employed for water and the PNIPAM structure is taken into account at the at. level. We identify the phys. factors driving or opposing the transition and evaluate their relative magnitudes and temp. dependences. The presence of PNIPAM generates an excluded vol. (EV) which is inaccessible to the centers of water mols. in the entire system. The presence of a water mol. also generates an EV for the other water mols. with the result that all of the water mols. are entropically correlated, causing water crowding. The globule state, where the EV is smaller and water crowding is less significant, is more favored in terms of the translational, configurational entropy of water. This effect always opposes the globule-to-coil transition. At low temps., however, this effect becomes significantly weaker, yielding to the factors driving it. The mechanism of the transition is phys. the same as that of cold denaturation of a protein.
  9. 9
    Graziano, G. On the temperature-induced coil to globule transition of poly-N-isopropylacrylamide in dilute aqueous solutions. Int. J. Biol. Macromol. 2000, 27, 89– 97,  DOI: 10.1016/S0141-8130(99)00122-1
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    On the temperature-induced coil to globule transition of poly-N-isopropylacrylamide in dilute aqueous solutions
    Graziano, G.
    International Journal of Biological Macromolecules (2000), 27 (1), 89-97CODEN: IJBMDR; ISSN:0141-8130. (Elsevier Science B.V.)
    Poly-N-isopropylacrylamide (PNIPAM) is a chem. isomer of poly-leucine, having the polar peptide group in the side-chain rather than in the backbone. It has been demonstrated exptl. that PNIPAM dissolved in aq. soln. undergoes a collapse transition from coil to globule on increasing temp. above the θ-point. By a careful reviewing of existing exptl. data, we emphasize that such coil to globule collapse has to be considered an intramol. first-order transition, analogous to the cold renaturation of small globular proteins. The main theor. approaches to the coil to globule collapse in homopolymers are discussed briefly, and a crit. comparison between the existing models is performed. We point out that, as a general result, the coil to globule collapse is expected to be a first-order transition for rigid and semi-rigid macromols. Finally, taking advantage of the analogy between the coil to globule collapse of PNIPAM and the cold renaturation of small globular proteins, we try to clarify some important and intriguing aspects of protein thermodn. This leads to the conclusion that the amphiphilic nature of polypeptide chain plays the fundamental role for the existence of two temp.-induced conformational transitions.
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    Tiktopulo, E. I.; Uversky, V. N.; Lushchik, V. B.; Klenin, S. I.; Bychkova, V. E.; Ptitsyn, O. B. Domain” Coil-Globule Transition in Homopolymers. Macromolecules 1995, 28, 7519– 7524,  DOI: 10.1021/ma00126a032
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    "Domain" Coil-Globule Transition in Homopolymers
    Tiktopulo, Elizaveta I.; Uversky, Vladimir N.; Lushchik, Vanda B.; Klenin, Stanislav I.; Bychkova, Valentina E.; Ptitsyn, Oleg B.
    Macromolecules (1995), 28 (22), 7519-24CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
    The temp.-induced coil-globule transition was studied in dil. aq. solns. (with 200 mg/L SDS) for different fractions of poly(N-isopropylacrylamide) (PNIPAM) and poly(N-isopropylmethacrylamide) (PNIPMAM) using scanning microcalorimetry, diffusion, and size-exclusion chromatog. (FPLC). Both these polymers undergo a coil-globule transition upon temp. increase. This transition is accompanied by cooperative heat absorption and a decrease of heat capacity, which makes it similar to the cold denaturation of globular proteins. The globule-coil transition is an all-or-none process only for the fractions with the lowest mol. wts. (∼10 × 103) while fractions of higher mol. wts. behave as if they consist of quasi-independent cooperative units, the domains. The no. of domains in a macromol. is proportional to the mol. wt. of the polymer. This suggests that the domain character of cooperative transitions in large proteins does not, in principle, need evolutionary-selected amino acid sequences but can occur even in homopolymers.
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    Fujishige, S.; Kubota, K.; Ando, I. Phase transition of aqueous solutions of poly(N-isopropylacrylamide) and poly(N-isopropylmethacrylamide). J. Phys. Chem. 1989, 93, 3311– 3313,  DOI: 10.1021/j100345a085
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    Phase transition of aqueous solutions of poly(N-isopropylacrylamide) and poly(N-isopropylmethacrylamide)
    Fujishige, Shouei; Kubota, K.; Ando, I.
    Journal of Physical Chemistry (1989), 93 (8), 3311-13CODEN: JPCHAX; ISSN:0022-3654.
    The coil-to-globule conformational transition of poly(N-isopropylacrylamide) in aq. solns. occurred at ∼32° as detected by optical measurements. This transition temp. was unaffected by the polymer mol. wt. ((5-840) × 104) or concn. (0.01-1 wt.%). The heat of transition was of the order 10 cal/g polymer. The conformational transitions of poly(N-isopropylmethacrylamide) and methylcellulose in aq. solns. shown for comparison.
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    Rosi, B. P.; Tavagnacco, L.; Comez, L.; Sassi, P.; Ricci, M.; Buratti, E.; Bertoldo, M.; Petrillo, C.; Zaccarelli, E.; Chiessi, E.; Corezzi, S. Thermoresponsivity of poly(N-isopropylacrylamide) microgels in water-trehalose solution and its relation to protein behavior. J. Colloid Interface Sci. 2021, 604, 705– 718,  DOI: 10.1016/j.jcis.2021.07.006
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    Thermoresponsivity of poly(N-isopropylacrylamide) microgels in water-trehalose solution and its relation to protein behavior
    Rosi, Benedetta Petra; Tavagnacco, Letizia; Comez, Lucia; Sassi, Paola; Ricci, Maria; Buratti, Elena; Bertoldo, Monica; Petrillo, Caterina; Zaccarelli, Emanuela; Chiessi, Ester; Corezzi, Silvia
    Journal of Colloid and Interface Science (2021), 604 (), 705-718CODEN: JCISA5; ISSN:0021-9797. (Elsevier B.V.)
    Additives are commonly used to tune macromol. conformational transitions. Among additives, trehalose is an excellent bioprotectant and among responsive polymers, PNIPAM is the most studied material. Nevertheless, their interaction mechanism so far has only been hinted without direct investigation, and, crucially, never elucidated in comparison to proteins. Detailed insights would help understand to what extent PNIPAM microgels can effectively be used as synthetic biomimetic materials, to reproduce and study, at the colloidal scale, isolated protein behavior and its sensitivity to interactions with specific cosolvents or cosolutes. The effect of trehalose on the swelling behavior of PNIPAM microgels was monitored by dynamic light scattering; Raman spectroscopy and mol. dynamics simulations were used to explore changes of solvation and dynamics across the swelling-deswelling transition at the mol. scale. Strongly hydrated trehalose mols. develop water-mediated interactions with PNIPAM microgels, thereby preserving polymer hydration below and above the transition while drastically inhibiting local motions of the polymer and of its hydration shell. Our study, for the first time, demonstrates that slowdown of dynamics and preferential exclusion are the principal mechanisms governing trehalose effect on PNIPAM microgels, at odds with preferential adsorption of alcs., but in full analogy with the behavior obsd. in trehalose-protein systems.
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    Yan, X.; Chu, Y.; Liu, B.; Ru, G.; Di, Y.; Feng, J. Dynamic Mechanism of Halide Salts on the Phase Transition of Protein Models Poly(N-isopropylacrylamide) and Poly(N,N-diethylacrylamide). Phys. Chem. Chem. Phys. 2020, 22, 12644– 12650,  DOI: 10.1039/D0CP01366H
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    Dynamic mechanism of halide salts on the phase transition of protein models, poly(N-isopropylacrylamide) and poly(N,N-diethylacrylamide)
    Yan, Xiaoshuang; Chu, Yueying; Liu, Biaolan; Ru, Geying; Di, Yi; Feng, Jiwen
    Physical Chemistry Chemical Physics (2020), 22 (22), 12644-12650CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)
    The effects of salts on protein systems are not yet fully understood. We investigated the ionic dynamics of three halide salts (NaI, NaBr, and NaCl) with two protein models, namely poly(N-isopropylacrylamide) (PNIPAM) and poly(N,N-diethylacrylamide) (PDEA), using multinuclear NMR, dispersion cor. d. functional theory (DFT-D) calcns. and dynamic light scattering (DLS) methods. The variation in ionic line-widths and chem. shifts induced by the polymers clearly illustrates that anions rather than cations interact directly with the polymers. From the variable temp. measurements of the NMR transverse relaxation rates of anions, which characterize the polymer-anion interaction intensities, the evolution behaviors of Cl-/Br-/I- during phase transitions are similar in each polymer system but differ between the two polymer systems. The NMR transverse relaxation rates of anions change synchronously with the phase transition of PNIPAM upon heating, but they drop rapidly and vanish about 3-4.5° before the phase transition of PDEA. By combining the DFT-D and DLS data, the relaxation results imply that anions escape from the interacting sites with PDEA prior to full polymer dehydration or collapse, which can be attributed to the lack of anion-NH interactions. The different dynamic evolutions of the anions in the PNIPAM and PDEA systems give us an important clue for understanding the micro-mechanism of protein folding in a complex salt aq. solvent.
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    Zhang, Y.; Furyk, S.; Bergbreiter, D. E.; Cremer, P. S. Specific Ion Effects on the Water Solubility of Macromolecules: PNIPAM and the Hofmeister Series. J. Am. Chem. Soc. 2005, 127, 14505– 14510,  DOI: 10.1021/ja0546424
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    Specific Ion Effects on the Water Solubility of Macromolecules: PNIPAM and the Hofmeister Series
    Zhang, Yanjie; Furyk, Steven; Bergbreiter, David E.; Cremer, Paul S.
    Journal of the American Chemical Society (2005), 127 (41), 14505-14510CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Aq. processes ranging from protein folding and enzyme turnover to colloidal ordering and macromol. pptn. are sensitive to the nature and concn. of the ions present in soln. Herein, the effect of a series of sodium salts on the lower crit. soln. temp. (LCST) of poly(N-isopropylacrylamide), PNIPAM, was investigated with a temp. gradient microfluidic device under a dark-field microscope. While the ability of a particular anion to lower the LCST generally followed the Hofmeister series, anal. of solvent isotope effects and of the changes in LCST with ion concn. and identity showed multiple mechanisms were at work. In solns. contg. sufficient concns. of strongly hydrated anions, the phase transition of PNIPAM was directly correlated with the hydration entropy of the anion. On the other hand, weakly hydrated anions were salted-out through surface tension effects and displayed improved hydration by direct ion binding.
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    Wu, J.; Wang, X.; Wang, Q.; Lou, Z.; Li, S.; Zhu, Y.; Qin, L.; Wei, H. Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes (II). Chem. Soc. Rev. 2019, 48, 1004– 1076,  DOI: 10.1039/C8CS00457A
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    Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes (II)
    Wu, Jiangjiexing; Wang, Xiaoyu; Wang, Quan; Lou, Zhangping; Li, Sirong; Zhu, Yunyao; Qin, Li; Wei, Hui
    Chemical Society Reviews (2019), 48 (4), 1004-1076CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    Nanozymes are nanomaterials with enzyme-like characteristics (Chem. Soc. Rev., 2013, 42, 6060-6093). They have been developed to address the limitations of natural enzymes and conventional artificial enzymes. Along with the significant advances in nanotechnol., biotechnol., catalysis science, and computational design, great progress has been achieved in the field of nanozymes since the publication of the above-mentioned comprehensive review in 2013. To highlight these achievements, this review first discusses the types of nanozymes and their representative nanomaterials, together with the corresponding catalytic mechanisms whenever available. Then, it summarizes various strategies for modulating the activity and selectivity of nanozymes. After that, the broad applications from biomedical anal. and imaging to theranostics and environmental protection are covered. Finally, the current challenges faced by nanozymes are outlined and the future directions for advancing nanozyme research are suggested. The current review can help researchers know well the current status of nanozymes and may catalyze breakthroughs in this field.
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    Xie, J.; Zhang, X.; Wang, H.; Zheng, H.; Huang, Y.; Xie, J. Analytical and environmental applications of nanoparticles as enzyme mimetics. Trends in Analytical Chemistry 2012, 39, 114– 129,  DOI: 10.1016/j.trac.2012.03.021
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    Analytical and environmental applications of nanoparticles as enzyme mimetics
    Xie, Jianxin; Zhang, Xiaodan; Wang, Hui; Zheng, Huzhi; Huang, Yuming; Xie, Jianxin
    TrAC, Trends in Analytical Chemistry (2012), 39 (), 114-129CODEN: TTAEDJ; ISSN:0165-9936. (Elsevier B. V.)
    A review. Recently, the intrinsic enzyme-like activity of nanoparticles (NPs) has become a growing area of interest. Compared with natural enzymes, these enzyme-like NPs are stable against denaturing, low in cost, and highly resistant to high concns. of substrate. These advantages make them promising in various applications. In this review, we focus on recent advances in NPs as enzyme mimetics and their anal. and environmental applications. We pay special attention to the enzyme-like activity of magnetic NPs, cerium-oxide NPs, noble-metal NPs, carbon and other nanomaterials.
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    Vial, L.; Dumy, P. Artificial enzyme-based biosensors. New J. Chem. 2009, 33, 939– 946,  DOI: 10.1039/b822966j
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    Artificial enzyme-based biosensors
    Vial, Laurent; Dumy, Pascal
    New Journal of Chemistry (2009), 33 (5), 939-946CODEN: NJCHE5; ISSN:1144-0546. (Royal Society of Chemistry)
    A review. During the last forty years, enzyme-based biosensors have had great success in the detection and quantification of various biol.-relevant mols. However, native enzymes can sometimes be costly, delicate to manipulate or simply absent for a particular analyte. Hence, artificial or synthetic enzymes could be a useful alternative to natural proteins for the conception of new biosensors, since they can be, a priori, designed in their entirety, as well as more robust, available, chem. malleable and cheap, in comparison with their natural analogs. In this Perspective, the authors will provide a snapshot of this emerging research field.
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    Halperin, A.; Kröger, M.; Winnik, F. M. Poly(N-isopropylacrylamide) Phase Diagrams: Fifty Years of Research. Angew. Chem., Int. Ed. 2015, 54, 15342– 15367,  DOI: 10.1002/anie.201506663
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    Poly(N-isopropylacrylamide) Phase Diagrams: Fifty Years of Research
    Halperin, Avraham; Kroeger, Martin; Winnik, Francoise M.
    Angewandte Chemie, International Edition (2015), 54 (51), 15342-15367CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. In 1968, Heskins and Guillet published the first systematic study of the phase diagram of poly(N-isopropylacrylamide) (PNIPAM), at the time a "young polymer" first synthesized in 1956. Since then, PNIPAM became the leading member of the growing families of thermoresponsive polymers and of stimuli-responsive, "smart" polymers in general. Its thermal response is unanimously attributed to its phase behavior. Yet, in spite of 50 years of research, a coherent quant. picture remains elusive. In this Review we survey the reported phase diagrams, discuss the differences and comment on theor. ideas regarding their possible origins. We aim to alert the PNIPAM community to open questions in this reputably mature domain.
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    Tavagnacco, L.; Zanatta, M.; Buratti, E.; Rosi, B.; Frick, B.; Natali, F.; Ollivier, J.; Chiessi, E.; Bertoldo, M.; Zaccarelli, E.; Orecchini, A. Proteinlike dynamical transition of hydrated polymer chains. Physical Review Research 2021, 3, 013191,  DOI: 10.1103/PhysRevResearch.3.013191
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    Proteinlike dynamical transition of hydrated polymer chains
    Tavagnacco, L.; Zanatta, M.; Buratti, E.; Rosi, B.; Frick, B.; Natali, F.; Ollivier, J.; Chiessi, E.; Bertoldo, M.; Zaccarelli, E.; Orecchini, A.
    Physical Review Research (2021), 3 (1), 013191CODEN: PRRHAI; ISSN:2643-1564. (American Physical Society)
    Combining elastic incoherent neutron scattering expts. at different resolns. with mol. dynamics simulations, we report the observation of a proteinlike dynamical transition in linear chains of poly(N-isopropylacrylamide). We identify the onset of the transition at a temp. Td of about 225 K. Due to a global fit procedure, we find quant. agreement between measured and calcd. polymer mean-squared displacements at all temps. and time resolns. Our results confirm the generality of the dynamical transition in macromol. systems in aq. environments, independently of the internal polymer topol.
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    Tavagnacco, L.; Chiessi, E.; Zanatta, M.; Orecchini, A.; Zaccarelli, E. Water–Polymer Coupling Induces a Dynamical Transition in Microgels. J. Phys. Chem. Lett. 2019, 10, 870– 876,  DOI: 10.1021/acs.jpclett.9b00190
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    Water-Polymer Coupling Induces a Dynamical Transition in Microgels
    Tavagnacco, Letizia; Chiessi, Ester; Zanatta, Marco; Orecchini, Andrea; Zaccarelli, Emanuela
    Journal of Physical Chemistry Letters (2019), 10 (4), 870-876CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
    The long debated protein dynamical transition was recently found also in nonbiol. macromols., such as poly-N-isopropylacrylamide (PNIPAM) microgels. Here, by using atomistic mol. dynamics simulations, we report a description of the mol. origin of the dynamical transition in these systems. We show that PNIPAM and water dynamics below the dynamical transition temp. Td are dominated by Me group rotations and hydrogen bonding, resp. By comparing with bulk water, we unambiguously identify PNIPAM-water hydrogen bonding as mainly responsible for the occurrence of the transition. The obsd. phenomenol. thus crucially depends on the water-macromol. coupling, being relevant to a wide class of hydrated systems, independently from the biol. function.
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    Zanatta, M.; Tavagnacco, L.; Buratti, E.; Bertoldo, M.; Natali, F.; Chiessi, E.; Orecchini, A.; Zaccarelli, E. Evidence of a low-temperature dynamical transition in concentrated microgels. Science Advances 2018, 4, eaat5895  DOI: 10.1126/sciadv.aat5895
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    Dynamical transition of myoglobin revealed by inelastic neutron scattering
    Doster, Wolfgang; Cusack, Stephen; Petry, Winfried
    Nature (London, United Kingdom) (1989), 337 (6209), 754-6CODEN: NATUAS; ISSN:0028-0836.
    The technique of inelastic neutron scattering was used to investigate at. motion in hydrated myoglobin over the temp. range 4-350 K and on the mol. dynamics timescale 0.1-100 ps. At temps. <180 K myoglobin behaves as a harmonic solid, with essentially only vibrational motion. Above 180 K there is a striking dynamic transition arising from the excitation of nonvibrational motion, which is interpreted as corresponding to torsional jumps between states of different energy, with a mean energy asymmetry of 12 kJ mol-1. This extra mobility is reflected in a strong temp. dependence of the mean-square at. displacements, a phenomenon previously obsd. specifically for the heme Fe by Moessbauer spectroscopy, but on a much slower timescale (10-7 s). It also correlates with a glass-like transition in the hydration shell of myoglobin and with the temp. dependence of ligand-binding rates at the heme Fe, as monitored by flash photolysis. The dynamical behavior found for myoglobin (and other globular proteins) suggests a coupling of fast local motions to slower collective motions, which is a characteristic feature of other dense glass-forming systems.
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    Schiró, G.; Weik, M. Role of hydration water in the onset of protein structural dynamics. J. Phys.: Condens. Matter 2019, 31, 463002,  DOI: 10.1088/1361-648X/ab388a
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    Role of hydration water in the onset of protein structural dynamics
    Schiro, Giorgio; Weik, Martin
    Journal of Physics: Condensed Matter (2019), 31 (46), 463002CODEN: JCOMEL; ISSN:0953-8984. (IOP Publishing Ltd.)
    A review. Proteins are the mol. workhorses in a living organism. Their 3D structures are animated by a multitude of equil. fluctuations and specific out-of-equil. motions that are required for proteins to be biol. active. When studied as a function of temp., functionally relevant dynamics are obsd. at and above the so-called protein dynamical transition (∼240 K) in hydrated, but not in dry proteins. In this review we present and discuss the main exptl. and computational results that provided evidence for the dynamical transition, with a focus on the role of hydration water dynamics in sustaining functional protein dynamics. The coupling and mutual influence of hydration water dynamics and protein dynamics are discussed and the hypotheses illustrated that have been put forward to explain the phys. origin of their onsets.
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    Cornicchi, E.; Capponi, S.; Marconi, M.; Onori, G.; Paciaroni, A. Temperature dependence of fast fluctuations in single- and double-stranded DNA molecules: a neutron scattering investigation. Philos. Mag. 2007, 87, 509– 515,  DOI: 10.1080/14786430600909022
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    Temperature dependence of fast fluctuations in single- and double-stranded DNA molecules: a neutron scattering investigation
    Cornicchi, E.; Capponi, S.; Marconi, M.; Onori, G.; Paciaroni, A.
    Philosophical Magazine (2007), 87 (3-5), 509-515CODEN: PMHABF; ISSN:1478-6435. (Taylor & Francis Ltd.)
    Using elastic neutron scattering measurements we have investigated the picosecond dynamics of dry and hydrated powders of DNA in the double-stranded (dsDNA) and single-stranded (ssDNA) state in the temp. range from 20 to 300 K. The extd. mean square displacements of DNA hydrogen atoms exhibit an onset of anharmonicity at around 100 K. The dynamics of the hydrated samples shows a further anharmonic contribution appearing at a temp. Td = 230-240 K. Such dynamical behavior is similar to the well-studied dynamical transition found in hydrated protein powders. The mean square displacements of dsDNA and ssDNA are practically superimposed in the whole temp. range for both dry and hydrated samples. This suggests that the DNA local mobility in the picosecond timescale does not depend on the single- or double-stranded conformation.
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    Cornicchi, E.; De Francesco, A.; Marconi, M.; Onori, G.; Paciaroni, A. A relationship between solvent viscosity and biomolecule picosecond thermal fluctuations. Chem. Phys. 2008, 345, 219– 223,  DOI: 10.1016/j.chemphys.2007.07.022
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    A relationship between solvent viscosity and biomolecule picosecond thermal fluctuations
    Cornicchi, E.; De Francesco, A.; Marconi, M.; Onori, G.; Paciaroni, A.
    Chemical Physics (2008), 345 (2-3), 219-223CODEN: CMPHC2; ISSN:0301-0104. (Elsevier B.V.)
    Through elastic neutron scattering measurements, we investigated the picosecond dynamics of DNA in the hydrated powder state or embedded in glycerol glassy matrix from 20 K to 300 K. We calcd. the relaxational contribution of the mean square displacements (MSD) of DNA hydrogen atoms. We found the existence of a linear relationship between the inverse of the biomol. relaxational MSD and the logarithm of the bulk viscosity of the surrounding environment. From the comparison with the case of lysozyme in the same environments, for which the validity of the relationship was already verified, possible differences and analogies concerning the biomol.-to-solvent dynamical coupling can be stressed.
  26. 26
    Caliskan, G.; Briber, R. M.; Thirumalai, D.; García-Sakai, V.; Woodson, S. A.; Sokolov, A. P. Dynamic Transition in tRNA is Solvent Induced. J. Am. Chem. Soc. 2006, 128, 32– 33,  DOI: 10.1021/ja056444i
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    Dynamic Transition in tRNA is Solvent Induced
    Caliskan, Gokhan; Briber, Robert M.; Thirumalai, D.; Garcia-Sakai, Victoria; Woodson, Sarah A.; Sokolov, Alexei P.
    Journal of the American Chemical Society (2006), 128 (1), 32-33CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Dynamics of tRNA was studied using neutron scattering spectroscopy. Despite vast differences in the architecture and backbone structure of proteins and RNA, hydrated tRNA undergoes the dynamic transition at the same temp. as hydrated lysozyme. The similarity of the dynamic transition in RNA and proteins supports the idea that it is solvent induced. Because tRNA essentially has no Me groups, the results also suggest that Me groups are not the main contributor of the dynamic transition in biol. macromols. However, they may explain strong differences in the dynamics of tRNA and lysozyme obsd. at low temps.
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    Roh, J.; Briber, R.; Damjanovic, A.; Thirumalai, D.; Woodson, S.; Sokolov, A. Dynamics of tRNA at Different Levels of Hydration. Biophys. J. 2009, 96, 2755– 2762,  DOI: 10.1016/j.bpj.2008.12.3895
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    Dynamics of tRNA at different levels of hydration
    Roh, J. H.; Briber, R. M.; Damjanovic, A.; Thirumalai, D.; Woodson, S. A.; Sokolov, A. P.
    Biophysical Journal (2009), 96 (7), 2755-2762CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    The influence of hydration on the nanosecond timescale dynamics of tRNA is investigated using neutron scattering spectroscopy. Unlike protein dynamics, the dynamics of tRNA is not affected by Me group rotation. This allows for a simpler anal. of the influence of hydration on the conformational motions in RNA. We find that hydration affects the dynamics of tRNA significantly more than that of lysozyme. Both the characteristic length scale and the timescale of the conformational motions in tRNA depend strongly on hydration. Even the characteristic temp. of the so-called "dynamical transition" appears to be hydration-dependent in tRNA. The amplitude of the conformational motions in fully hydrated tRNA is almost twice as large as in hydrated lysozyme. We ascribe these differences to a more open and flexible structure of hydrated RNA, and to a larger fraction and different nature of hydrophilic sites. The latter leads to a higher d. of water that makes the biomol. more flexible. All-atom mol.-dynamics simulations are used to show that the extent of hydration is greater in tRNA than in lysozyme. We propose that water acts as a "lubricant" in facilitating enhanced motion in solvated RNA mols.
  28. 28
    Capaccioli, S.; Ngai, K. L.; Ancherbak, S.; Paciaroni, A. Evidence of Coexistence of Change of Caged Dynamics at Tg and the Dynamic Transition at Td in Solvated Proteins. J. Phys. Chem. B 2012, 116, 1745– 1757,  DOI: 10.1021/jp2057892
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    Evidence of Coexistence of Change of Caged Dynamics at Tg and the Dynamic Transition at Td in Solvated Proteins
    Capaccioli, S.; Ngai, K. L.; Ancherbak, S.; Paciaroni, A.
    Journal of Physical Chemistry B (2012), 116 (6), 1745-1757CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
    Moessbauer spectroscopy and neutron scattering measurements on proteins embedded in solvents including water and aq. mixts. have emphasized the observation of the distinctive temp. dependence of the at. mean square displacements, 〈U2〉, commonly referred to as the dynamic transition at some temp. Td. At low temps., 〈U2〉 increases slowly, but it assumes stronger temp. dependence after crossing Td, which depends on the time/frequency resoln. of the spectrometer. Various authors have made connection of the dynamics of solvated proteins, including the dynamic transition to that of glass-forming substances. Notwithstanding, no connection is made to the similar change of temp. dependence of 〈U2〉 obtained by quasielastic neutron scattering when crossing the glass transition temp. Tg, generally obsd. in inorg., org., and polymeric glass-formers. Evidences are presented here to show that such a change of the temp. dependence of 〈U2〉 from neutron scattering at Tg is present in hydrated or solvated proteins, as well as in the solvent used, unsurprisingly since the latter is just another org. glass-former. If unaware of the existence of such a crossover of 〈U2〉 at Tg, and if present, it can be mistaken as the dynamic transition at Td with the ill consequences of underestimating Td by the lower value Tg and confusing the identification of the origin of the dynamic transition. The 〈U2〉 obtained by neutron scattering at not so low temps. has contributions from the dissipation of mols. while caged by the anharmonic intermol. potential at times before dissoln. of cages by the onset of the Johari-Goldstein β-relaxation or of the merged α-β relaxation. The universal change of 〈U2〉 at Tg of glass-formers, independent of the spectrometer resoln., had been rationalized by sensitivity to change in vol. and entropy of the dissipation of the caged mols. and its contribution to 〈U2〉. The same rationalization applies to hydrated and solvated proteins for the obsd. change of 〈U2〉 at Tg.
  29. 29
    Ngai, K.; Capaccioli, S.; Paciaroni, A. Dynamics of hydrated proteins and bio-protectants: Caged dynamics, β-relaxation, and α-relaxation. Biochimica et Biophysica Acta (BBA) - General Subjects 2017, 1861, 3553– 3563,  DOI: 10.1016/j.bbagen.2016.04.027
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    Chen, S.-H.; Liu, L.; Fratini, E.; Baglioni, P.; Faraone, A.; Mamontov, E. Observation of fragile-to-strong dynamic crossover in protein hydration water. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 9012– 9016,  DOI: 10.1073/pnas.0602474103
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    Observation of fragile-to-strong dynamic crossover in protein hydration water
    Chen, S.-H.; Liu, L.; Fratini, E.; Baglioni, P.; Faraone, A.; Mamontov, E.
    Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (24), 9012-9016CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    At low temps., proteins exist in a glassy state, a state that has no conformational flexibility and shows no biol. functions. In a hydrated protein, at temps. ≥220 K, this flexibility is restored, and the protein is able to sample more conformational substates, thus becoming biol. functional. This "dynamical" transition of protein is believed to be triggered by its strong coupling with the hydration water, which also shows a similar dynamic transition. Here we demonstrate exptl. that this sudden switch in dynamic behavior of the hydration water on lysozyme occurs precisely at 220 K and can be described as a fragile-to-strong dynamic crossover. At the fragile-to-strong dynamic crossover (FSC), the structure of hydration water makes a transition from predominantly high-d. (more fluid state) to low-d. (less fluid state) forms derived from the existence of the second crit. point at an elevated pressure.
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    Wood, K.; Frölich, A.; Paciaroni, A.; Moulin, M.; Härtlein, M.; Zaccai, G.; Tobias, D. J.; Weik, M. Coincidence of Dynamical Transitions in a Soluble Protein and Its Hydration Water: Direct Measurements by Neutron Scattering and MD Simulations. J. Am. Chem. Soc. 2008, 130, 4586– 4587,  DOI: 10.1021/ja710526r
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    Coincidence of Dynamical Transitions in a Soluble Protein and Its Hydration Water: Direct Measurements by Neutron Scattering and MD Simulations
    Wood, Kathleen; Froelich, Andreas; Paciaroni, Alessandro; Moulin, Martine; Haertlein, Michael; Zaccai, Giuseppe; Tobias, Douglas J.; Weik, Martin
    Journal of the American Chemical Society (2008), 130 (14), 4586-4587CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The coupling between protein dynamics and hydration-water dynamics was assessed by perdeuteration, temp.-dependent neutron scattering, and mol. dynamics simulations. Mean square displacements of water and protein motions both show a broad transition at 220 K and are thus coupled. In particular, the protein dynamical transition appears to be driven by the onset of hydration-water translational motion.
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    Schiró, G.; Vetri, V.; Frick, B.; Militello, V.; Leone, M.; Cupane, A. Neutron Scattering Reveals Enhanced Protein Dynamics in Concanavalin A Amyloid Fibrils. J. Phys. Chem. Lett. 2012, 3, 992– 996,  DOI: 10.1021/jz300082x
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    Neutron scattering reveals enhanced protein dynamics in concanavalin A amyloid fibrils
    Schiro, Giorgio; Vetri, Valeria; Frick, Bernhard; Militello, Valeria; Leone, Maurizio; Cupane, Antonio
    Journal of Physical Chemistry Letters (2012), 3 (8), 992-996CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
    Protein aggregation is one of the most challenging topics in life sciences, and it is implicated in several human pathologies. The nature and the role of toxic species is highly debated, with amyloid fibrils being among the most relevant species for their peculiar structural and functional properties. Protein dynamics and in particular the ability to fluctuate through a large no. of conformational substates are closely related to protein function. Here, the authors focus on amyloid fibril dynamics, and, to their knowledge, it is the 1st neutron scattering study on a protein (Con A) isolated in its fibril state. The results revealed enhanced at. fluctuations in amyloid fibrils and indicated that the protein was "softer" in the fibril state with respect to the native and amorphous aggregate states. The authors discuss this finding in terms of a structural interpretation and suggest that the paradigm ordered structure ↔ lower flexibility can be questioned when considering the local fast side-chain protein dynamics.
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    Schiró, G.; Caronna, C.; Natali, F.; Koza, M. M.; Cupane, A. The “Protein Dynamical Transition” Does Not Require the Protein Polypeptide Chain. J. Phys. Chem. Lett. 2011, 2, 2275– 2279,  DOI: 10.1021/jz200797g
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    The "protein dynamical transition" does not require the protein polypeptide chain
    Schiro, Giorgio; Caronna, Chiara; Natali, Francesca; Koza, M. Marek; Cupane, Antonio
    Journal of Physical Chemistry Letters (2011), 2 (18), 2275-2279CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
    The authors provide exptl. evidence that the main features of protein dynamics revealed by neutron scattering, i.e., the "protein dynamical transition" and the "boson peak", do not require the protein polypeptide chain. The authors show that a rapid increase of H atom fluctuations at ∼220 K, analogous to the one obsd. in hydrated myoglobin powders, was also obsd. in a mixt. of hydrated amino acids with the chem. compn. of myoglobin but lacking the polypeptide chain; in agreement with the protein behavior, the transition was abolished in the dry mixt. Further, an excess of low-frequency vibrational modes at ∼3 meV, typically obsd. in protein powders, was also obsd. in the authors' mixt. These results confirmed that the dynamical transition is a water-driven onset and indicated that it mainly involves the amino acid side chains. Taking together the present data and recent results on the dynamics of a protein in denatured conformation and on the activity of dehydrated proteins, it could be concluded that the "protein dynamical transition" is neither a necessary nor a sufficient condition for active protein conformation and function.
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    Niessen, K. A.; Xu, M.; Paciaroni, A.; Orecchini, A.; Snell, E. H.; Markelz, A. G. Moving in the Right Direction: Protein Vibrations Steering Function. Biophys. J. 2017, 112, 933– 942,  DOI: 10.1016/j.bpj.2016.12.049
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    Moving in the Right Direction: Protein Vibrations Steering Function
    Niessen, Katherine A.; Xu, Mengyang; Paciaroni, Alessandro; Orecchini, Andrea; Snell, Edward H.; Markelz, Andrea G.
    Biophysical Journal (2017), 112 (5), 933-942CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    Nearly all protein functions require structural change, such as enzymes clamping onto substrates, and ion channels opening and closing. These motions are a target for possible new therapies; however, the control mechanisms are under debate. Calcns. have indicated protein vibrations enable structural change. However, previous measurements found these vibrations only weakly depend on the functional state. By using the novel technique of anisotropic terahertz microscopy (ATM), we find that there is a dramatic change to the vibrational directionality with inhibitor (3NAG) binding to lysozyme, whereas the vibrational energy distribution, as measured by neutron inelastic scattering, is only slightly altered. The anisotropic terahertz measurements provide unique access to the directionality of the intramol. vibrations, and immediately resolve the inconsistency between calcns. and previous measurements, which were only sensitive to the energy distribution. The biol. importance of the vibrational directions vs. the energy distribution is revealed by our calcns. comparing wild-type lysozyme with a higher catalytic rate double deletion mutant. The vibrational energy distribution is identical, but the more efficient mutant shows an obvious reorientation of motions. These results show that it is essential to characterize the directionality of motion to understand and control protein dynamics to optimize or inhibit function.
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    Henzler-Wildman, K.; Kern, D. Dynamic personalities of proteins. Nature 2007, 450, 964– 972,  DOI: 10.1038/nature06522
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    Dynamic personalities of proteins
    Henzler-Wildman, Katherine; Kern, Dorothee
    Nature (London, United Kingdom) (2007), 450 (7172), 964-972CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    A review. Because proteins are central to cellular function, researchers have sought to uncover the secrets of how these complex macromols. execute such a fascinating variety of functions. Although static structures are known for many proteins, the functions of proteins are governed ultimately by their dynamic character (or 'personality'). The dream is to 'watch' proteins in action in real time at at. resoln. This requires the addn. of a fourth dimension, time, to structural biol. so that the positions in space and time of all atoms in a protein can be described in detail.
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    Fenimore, P. W.; Frauenfelder, H.; McMahon, B. H.; Young, R. D. Bulk-solvent and hydration-shell fluctuations, similar to α- and β-fluctuations in glasses, control protein motions and functions. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 14408– 14413,  DOI: 10.1073/pnas.0405573101
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    Bulk-solvent and hydration-shell fluctuations, similar to α- and β-fluctuations in glasses, control protein motions and functions
    Fenimore, P. W.; Frauenfelder, Hans; McMahon, B. H.; Young, R. D.
    Proceedings of the National Academy of Sciences of the United States of America (2004), 101 (40), 14408-14413CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The concept that proteins exist in numerous different conformations or conformational substates, described by an energy landscape, is now accepted, but the dynamics is incompletely explored. We have previously shown that large-scale protein motions, such as the exit of a ligand from the protein interior, follow the dielec. fluctuations in the bulk solvent. Here, we demonstrate, by using mean-square displacements (msd) from Moessbauer and neutron-scattering expts., that fluctuations in the hydration shell control fast fluctuations in the protein. We call the first type solvent-slaved or α-fluctuations and the second type hydration-shell-coupled or β-fluctuations. Solvent-slaved motions are similar to the α-fluctuations in glasses. Their temp. dependence can be approximated by a Vogel-Tammann-Fulcher relation and they are absent in a solid environment. Hydration-shell-coupled fluctuations are similar to the β-relaxation in glasses. They can be approximated by a Ferry or an Arrhenius relation, are much reduced or absent in dehydrated proteins, and occur in hydrated proteins even if embedded in a solid. They can be responsible for internal processes such as the migration of ligands within myoglobin. The existence of two functionally important fluctuations in proteins, one slaved to bulk motions and the other coupled to hydration-shell fluctuations, implies that the environment can control protein functions through different avenues and that no real protein transition occurs at ≈200 K. The large no. of conformational substates is essential; proteins cannot function without this reservoir of entropy, which resides mainly in the hydration shell.
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    Rasmussen, B. F.; Stock, A. M.; Ringe, D.; Petsko, G. A. Crystalline Ribonuclease A Loses Function Below the Dynamical Transition at 220 K. Nature 1992, 357, 423– 424,  DOI: 10.1038/357423a0
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    Crystalline ribonuclease A loses function below the dynamical transition at 220 K
    Rasmussen, Bjarne F.; Stock, Ann M.; Ringe, Dagmar; Petsko, Gregory A.
    Nature (London, United Kingdom) (1992), 357 (6377), 423-4CODEN: NATUAS; ISSN:0028-0836.
    When the dynamic properties of many different proteins are plotted as a function of temp., biphasic behavior is obsd., with a broad transition centered around 220 K. Mol. dynamics simulations over a range of temps. also exhibit a transition at about 220 K. Here, high-resoln. x-ray diffraction was used to show that cryst. RNase A does not bind substrate or inhibitor at 212 K but will bind either rapidly at 228 K. Once bound at the higher temp., inhibitor cannot be washed off after the enzyme is cooled to below the transition temp. These results suggest that enzyme flexibility is required for catalytic function.
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    Réat, V.; Patzelt, H.; Ferrand, M.; Pfister, C.; Oesterhelt, D.; Zaccai, G. Dynamics of different functional parts of bacteriorhodopsin: H-2H labeling and neutron scattering. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 4970– 4975,  DOI: 10.1073/pnas.95.9.4970
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    Dynamics of different functional parts of bacteriorhodopsin: H-2H labeling and neutron scattering
    Reat, Valerie; Patzelt, Heiko; Ferrand, Michel; Pfister, Claude; Oesterhelt, Dieter; Zaccai, Giuseppe
    Proceedings of the National Academy of Sciences of the United States of America (1998), 95 (9), 4970-4975CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    We show that dynamics of specific amino acids within a protein can be characterized by neutron spectroscopy and hydrogen-deuterium labeling, and we present data on the motions of a selected set of groups within bacteriorhodopsin (BR), the retinal-based proton pump in the purple membrane of halophilic Archaea. Elastic incoherent neutron scattering expts. allow the definition of motions in the nano- to picosecond time scale and have revealed a dynamical transition from a harmonic to a softer, anharmonic at. fluctuation regime in the global behavior of proteins. Biol. activity in proteins is correlated with this transition, suggesting that flexibility is required for function. Elastic incoherent neutron scattering is dominated by H atom scattering, and to study the dynamics of a selected part of BR, fully deuterated purple membrane with BR contg. H-retinal, H-tryptophan, and H-methionine was prepd. biosynthetically in Halobacterium salinarum. These amino acids cluster in the functional center of the protein. In contrast to the protein globally, the thermal motions of the labeled atoms were found to be shielded from solvent melting effects at 260 K. Above this temp., the labeled groups appear as more rigid than the rest of the protein, with a significantly smaller mean square amplitude of motion. These exptl. results quantify the dynamical heterogeneity of BR (which meets the functional requirements of global flexibility), on the one hand, to allow large conformational changes in the mol. and of a more rigid region in the protein, on the other, to control stereo-specific selection of retinal conformations.
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    Mensink, M. A.; Frijlink, H. W.; van der Voort Maarschalk, K.; Hinrichs, W. L. How sugars protect proteins in the solid state and during drying (review): Mechanisms of stabilization in relation to stress conditions. Eur. J. Pharm. Biopharm. 2017, 114, 288– 295,  DOI: 10.1016/j.ejpb.2017.01.024
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    How sugars protect proteins in the solid state and during drying (review): Mechanisms of stabilization in relation to stress conditions
    Mensink, Maarten A.; Frijlink, Henderik W.; van der Voort Maarschalk, Kees; Hinrichs, Wouter L. J.
    European Journal of Pharmaceutics and Biopharmaceutics (2017), 114 (), 288-295CODEN: EJPBEL; ISSN:0939-6411. (Elsevier B.V.)
    This review aims to provide an overview of current knowledge on stabilization of proteins by sugars in the solid state in relation to stress conditions commonly encountered during drying and storage. First protein degrdn. mechanisms in the solid state (i.e. phys. and chem. degrdn. routes) and traditional theories regarding protein stabilization (vitrification and water replacement hypotheses) will be briefly discussed. Secondly, refinements to these theories, such as theories focusing on local mobility and protein-sugar packing d., are reviewed in relationship to the traditional theories and their analogies are discussed. The last section relates these mechanistic insights to the stress conditions against which these sugars are used to provide protection (i.e. drying, temp., and moisture). In summary sugars should be able to adequately form interactions with the protein during drying, thereby maintaining it in its native conformation and reducing both local and global mobility during storage. Generally smaller sugars (disaccharides) are better at forming these interactions and reducing local mobility as they are less inhibited by steric hindrance, while larger sugars can reduce global mobility more efficiently. The principles outlined here can aid in choosing a suitable sugar as stabilizer depending on the protein, formulation and storage condition-specific dominant route of degrdn.
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    Cicerone, M. T.; Pikal, M. J.; Qian, K. K. Stabilization of proteins in solid form. Adv. Drug Delivery Rev. 2015, 93, 14– 24,  DOI: 10.1016/j.addr.2015.05.006
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    Stabilization of proteins in solid form
    Cicerone, Marcus T.; Pikal, Michael J.; Qian, Ken K.
    Advanced Drug Delivery Reviews (2015), 93 (), 14-24CODEN: ADDREP; ISSN:0169-409X. (Elsevier B.V.)
    Immunogenicity of aggregated or otherwise degraded protein delivered from depots or other biopharmaceutical products is an increasing concern, and the ability to deliver stable, active protein is of central importance. We review characterization approaches for solid protein dosage forms with respect to metrics that are intended to be predictive of protein stability against aggregation and other degrdn. processes. Each of these approaches is ultimately motivated by hypothetical connections between protein stability and the material property being measured. We critically evaluate correlations between these properties and stability outcomes, and use these evaluations to revise the currently standing hypotheses. Based on this we provide simple phys. principles that are necessary (and possibly sufficient) for generating solid delivery vehicles with stable protein loads. Essentially, proteins should be strongly coupled (typically through H-bonds) to the bulk regions of a phase-homogeneous matrix with suppressed β relaxation. We also provide a framework for reliable characterization of solid protein forms with respect to stability.
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    Manning, M.; Chou, D.; Murphy, B.; Payne, R.; Katayama, D. Stability of Protein Pharmaceuticals: An Update. Pharm. Res. 2010, 27, 544– 575,  DOI: 10.1007/s11095-009-0045-6
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    Stability of protein pharmaceuticals: an update
    Manning Mark Cornell; Chou Danny K; Murphy Brian M; Payne Robert W; Katayama Derrick S
    Pharmaceutical research (2010), 27 (4), 544-75 ISSN:.
    In 1989, Manning, Patel, and Borchardt wrote a review of protein stability (Manning et al., Pharm. Res. 6:903-918, 1989), which has been widely referenced ever since. At the time, recombinant protein therapy was still in its infancy. This review summarizes the advances that have been made since then regarding protein stabilization and formulation. In addition to a discussion of the current understanding of chemical and physical instability, sections are included on stabilization in aqueous solution and the dried state, the use of chemical modification and mutagenesis to improve stability, and the interrelationship between chemical and physical instability.
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    Cordone, L.; Ferrand, M.; Vitrano, E.; Zaccai, G. Harmonic Behavior of Trehalose-Coated Carbon-Monoxy-Myoglobin at High Temperature. Biophys. J. 1999, 76, 1043– 1047,  DOI: 10.1016/S0006-3495(99)77269-3
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    Harmonic behavior of trehalose-coated carbon-monoxy-myoglobin at high temperature
    Cordone, Lorenzo; Ferrand, Michel; Vitrano, Eugenio; Zaccai, Giuseppe
    Biophysical Journal (1999), 76 (2), 1043-1047CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    Embedding biostructures in saccharide glasses protects them against extreme dehydration and/or exposure to very high temp. Among the saccharides, trehalose appears to be the most effective bioprotectant. In this paper we report on the low-frequency dynamics of carbon monoxy myoglobin in an extremely dry trehalose glass measured by neutron spectroscopy. Under these conditions, the mean square displacements and the d. of state function are those of a harmonic solid, up to room temp., in contrast to D2O-hydrated myoglobin, in which a dynamical transition to a nonharmonic regime has been obsd. at ∼180 K (Doster et al., 1989. Nature. 337:754-756). The protective effect of trehalose is correlated, therefore, with a trapping of the protein in a harmonic potential, even at relatively high temp.
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    Cornicchi, E.; Marconi, M.; Onori, G.; Paciaroni, A. Controlling the Protein Dynamical Transition with Sugar-Based Bioprotectant Matrices: A Neutron Scattering Study. Biophys. J. 2006, 91, 289– 297,  DOI: 10.1529/biophysj.106.081752
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    Controlling the protein dynamical transition with sugar-based bioprotectant matrices: a neutron scattering study
    Cornicchi, E.; Marconi, M.; Onori, G.; Paciaroni, A.
    Biophysical Journal (2006), 91 (1), 289-297CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    Through elastic neutron scattering we measured the mean-square displacements (MSDs) of the hydrogen atoms of lysozyme embedded in a glucose-water glassy matrix as a function of the temp. and at various water contents. The elastic intensity of all the samples has been interpreted in terms of the double-well model in the whole temp. range. The dry sample shows an onset of anharmonicity at ∼100 K, which can be attributed to the activation of Me group reorientations. Such a protein intrinsic dynamics is decoupled from the external environment on the whole investigated temp. range. In the hydrated samples an addnl. and larger anharmonic contribution is provided by the protein dynamical transition, which appears at a higher temp. Td. As hydration increases the coupling between the protein internal dynamics and the surrounding matrix relaxations becomes more effective. The behavior of Td that, as a function of the water content, diminishes by ∼60 K, supports the picture of the protein dynamics as driven by solvent relaxations. A possible connection between the protein dynamical response vs. T and the thermal stability in glucose-water bioprotectant matrixes is proposed.
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    Paciaroni, A.; Cinelli, S.; Onori, G. Effect of the Environment on the Protein Dynamical Transition: A Neutron Scattering Study. Biophys. J. 2002, 83, 1157– 1164,  DOI: 10.1016/S0006-3495(02)75239-9
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    Effect of the environment on the protein dynamical transition: a neutron scattering study
    Paciaroni, Alessandro; Cinelli, Stefania; Onori, Giuseppe
    Biophysical Journal (2002), 83 (2), 1157-1164CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    The authors performed an elastic neutron scattering investigation of the mol. dynamics of lysozyme solvated in glycerol, at different water contents h (grams of water/g of lysozyme). The marked nonGaussian behavior of the elastic intensity was studied in a wide exptl. momentum transfer range, as a function of the temp. The internal dynamics is well described in terms of the double-well jump model. At low temp., the protein total mean square displacements exhibit an almost linear harmonic trend irresp. of the hydration level, whereas at the temp. Td a clear changeover toward an anharmonic regime marks a protein dynamical transition. The decrease of Td from ∼238 K to ∼195 K as a function of h is reminiscent of that found in the glass transition temp. of aq. solns. of glycerol, thus suggesting that the protein internal dynamics as a whole is slave to the environment properties. Both Td and the total mean square displacements indicate that the protein flexibility strongly rises between 0.1 and 0.2h. This hydration-dependent dynamical activation, which is similar to that of hydrated lysozyme powders, is related to the specific interplay of the protein with the surrounding water and glycerol mols.
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    Katava, M.; Stirnemann, G.; Zanatta, M.; Capaccioli, S.; Pachetti, M.; Ngai, K. L.; Sterpone, F.; Paciaroni, A. Critical structural fluctuations of proteins upon thermal unfolding challenge the Lindemann criterion. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 9361– 9366,  DOI: 10.1073/pnas.1707357114
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    Critical structural fluctuations of proteins upon thermal unfolding challenge the Lindemann criterion
    Katava, Marina; Stirnemann, Guillaume; Zanatta, Marco; Capaccioli, Simone; Pachetti, Maria; Ngai, K. L.; Sterpone, Fabio; Paciaroni, Alessandro
    Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (35), 9361-9366CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Internal subnanosecond timescale motions are key for the function of proteins, and are coupled to the surrounding solvent environment. These fast fluctuations guide protein conformational changes, yet their role for protein stability, and for unfolding, remains elusive. Here, in analogy with the Lindemann criterion for the melting of solids, we demonstrate a common scaling of structural fluctuations of lysozyme protein embedded in different environments as the thermal unfolding transition is approached. By combining elastic incoherent neutron scattering and advanced mol. simulations, we show that, although different solvents modify the protein melting temp., a unique dynamical regime is attained in proximity of thermal unfolding in all solvents that we tested. This solvation shell-independent dynamical regime arises from an equiv. sampling of the energy landscape at the resp. melting temps. Thus, we propose that a threshold for the conformational entropy provided by structural fluctuations of proteins exists, beyond which thermal unfolding is triggered.
  46. 46
    Marconi, M.; De Francesco, A.; Cornicchi, E.; Onori, G.; Paciaroni, A. Hydration and temperature dependent dynamics of lysozyme in glucose–water matrices. A neutron scattering study. Chem. Phys. 2005, 317, 274– 281,  DOI: 10.1016/j.chemphys.2005.06.027
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    Hydration and temperature dependent dynamics of lysozyme in glucose-water matrices. A neutron scattering study
    Marconi, M.; De Francesco, A.; Cornicchi, E.; Onori, G.; Paciaroni, A.
    Chemical Physics (2005), 317 (2-3), 274-281CODEN: CMPHC2; ISSN:0301-0104. (Elsevier B.V.)
    Through a neutron scattering expt., we studied the dynamics of lysozyme embedded in glucose-water matrixes. Two distinct quasielastic contributions, corresponding to motions in the sub- and picosecond timescales, are revealed after the subtraction of the inelastic intensity, as estd. at low temp. Their characteristic times are hydration and temp. independent, and quite similar to those revealed when the environment around the biomol. surface is pure water or glycerol. The momentum transfer dependence of each quasielastic signal has been interpreted in terms of the rotational diffusion model. The faster component corresponds to a more localized rotational motion on a radius of 1 Å, while the slower contribution is assocd. to a larger radius of 1.9 Å. The behavior of the fraction of moving protein hydrogen atoms as a function of temp. and hydration suggests that the protein undergoes a dynamical transition just at conditions corresponding to the dynamical onset of the enclosing matrix.
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    Paciaroni, A.; Cornicchi, E.; De Francesco, A.; Marconi, M.; Onori, G. Conditioning action of the environment on the protein dynamics studied through elastic neutron scattering. Eur. Biophys. J. 2006, 35, 591– 599,  DOI: 10.1007/s00249-006-0073-7
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    Conditioning action of the environment on the protein dynamics studied through elastic neutron scattering
    Paciaroni, A.; Cornicchi, E.; De Francesco, A.; Marconi, M.; Onori, G.
    European Biophysics Journal (2006), 35 (7), 591-599CODEN: EBJOE8; ISSN:0175-7571. (Springer)
    The dynamics of lysozyme in the picosecond timescale was studied when it is in dry and hydrated powder form and when it is embedded in glycerol, glycerol-water, glucose and glucose-water matrixes. The investigation was undertaken through elastic neutron scattering technique on the backscattering spectrometer IN13. The dynamics of dry powder and embedded-in-glucose lysozyme can be considered purely vibrational up to 100 K, where the onset of an anharmonic contribution takes place. This contribution can be attributed to the activation of Me group reorientations and is described with an Arrhenius trend. An addnl. source of anharmonic dynamics appears at higher temps. for lysozyme in hydrated powders and embedded in glycerol, glycerol-water and glucose-water matrixes. This second process, also represented with an Arrhenius trend, corresponds to the so-called protein dynamical transition. Both the temp. where such a transition takes place and the magnitude of the protein mean square displacements depend on the environment. The dynamical response of the protein to temp. is put in relationship with its thermal stability.
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    Tsai, A. M.; Neumann, D. A.; Bell, L. N. Molecular Dynamics of Solid-State Lysozyme as Affected by Glycerol and Water: A Neutron Scattering Study. Biophys. J. 2000, 79, 2728– 2732,  DOI: 10.1016/S0006-3495(00)76511-8
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    Molecular dynamics of solid-state lysozyme as affected by glycerol and water: a neutron scattering study
    Tsai, Amos M.; Neumann, Dan A.; Bell, Leonard N.
    Biophysical Journal (2000), 79 (5), 2728-2732CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    Glycerol has previously been shown to lower the heat denaturation temp. (Tm) of dehydrated lysozyme while elevating the Tm of hydrated lysozyme. Here, the authors report an in situ elastic neutron scattering study of the effect of glycerol and hydration on the internal dynamics of lysozyme powder. Anharmonic motions assocd. with structural relaxation processes were not detected for dehydrated lysozyme in the temp. range of 40-450K. Dehydrated lysozyme was previously found to have the highest Tm. Upon the addn. of glycerol or water, anharmonicity was recovered above a dynamic transition temp. (Td), which may contribute to the redn. of Tm values for dehydrated lysozyme in the presence of glycerol. The greatest degree of anharmonicity, as well as the lowest Td, was obsd. for lysozyme solvated with water. Hydrated lysozyme was also previously found to have the lowest Tm. In the regime above the Td, larger amts. of glycerol led to a higher rate of change in anharmonic motions as a function of temp., rendering the material more heat-labile. Below the Td, where harmonic motions dominate, the addn. of glycerol resulted in a lower amplitude of motions, correlating with a stabilizing effect of glycerol on the protein.
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    Tsai, A. M.; Udovic, T. J.; Neumann, D. A. The Inverse Relationship between Protein Dynamics and Thermal Stability. Biophys. J. 2001, 81, 2339– 2343,  DOI: 10.1016/S0006-3495(01)75880-8
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    The inverse relationship between protein dynamics and thermal stability
    Tsai, Amos M.; Udovic, Terrence J.; Neumann, Dan A.
    Biophysical Journal (2001), 81 (4), 2339-2343CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    Protein powders that are dehydrated or mixed with a glassy compd. are known to have improved thermal stability. We present elastic and quasielastic neutron scattering measurements of the global dynamics of lysozyme and RNase A powders. In the absence of solvation water, both protein powders exhibit largely harmonic motions on the timescale of the measurements. Upon partial hydration, quasielastic scattering indicative of relaxational processes appears at sufficiently high temp. When the scattering spectrum are analyzed with the Kohlrausch-Williams-Watts formalism, the exponent β decreases with increasing temp., suggesting that multiple relaxation modes are emerging. When lysozyme was mixed with glycerol, its β values were higher than the hydrated sample at comparable temps., reflecting the viscosity and stabilizing effects of glycerol.
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    Weng, L.; Stott, S. L.; Toner, M. Exploring Dynamics and Structure of Biomolecules, Cryoprotectants, and Water Using Molecular Dynamics Simulations: Implications for Biostabilization and Biopreservation. Annu. Rev. Biomed. Eng. 2019, 21, 1– 31,  DOI: 10.1146/annurev-bioeng-060418-052130
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    Exploring Dynamics and Structure of Biomolecules, Cryoprotectants, and Water Using Molecular Dynamics Simulations: Implications for Biostabilization and Biopreservation
    Weng, Lindong; Stott, Shannon L.; Toner, Mehmet
    Annual Review of Biomedical Engineering (2019), 21 (), 1-31CODEN: ARBEF7; ISSN:1523-9829. (Annual Reviews)
    Successful stabilization and preservation of biol. materials often utilize low temps. and dehydration to arrest mol. motion. Cryoprotectants are routinely employed to help the biol. entities survive the physicochem. and mech. stresses induced by cold or dryness. Mol. interactions between biomols., cryoprotectants, and water fundamentally det. the outcomes of preservation. The optimization of assays using the empirical approach is often limited in structural and temporal resoln., whereas classical mol. dynamics simulations can provide a cost-effective glimpse into the at.-level structure and interaction of individual mols. that dictate macroscopic behavior. Computational research on biomols., cryoprotectants, and water has provided invaluable insights into the development of new cryoprotectants and the optimization of preservation methods. We describe the rapidly evolving state of the art of mol. simulations of these complex systems, summarize the mol.-scale protective and stabilizing mechanisms, and discuss the challenges that motivate continued innovation in this field.
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    Cicerone, M. T.; Soles, C. L. Fast Dynamics and Stabilization of Proteins: Binary Glasses of Trehalose and Glycerol. Biophys. J. 2004, 86, 3836– 3845,  DOI: 10.1529/biophysj.103.035519
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    Fast dynamics and stabilization of proteins: Binary glasses of trehalose and glycerol
    Cicerone, Marcus T.; Soles, Christopher L.
    Biophysical Journal (2004), 86 (6), 3836-3845CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    We present elastic and inelastic incoherent neutron scattering data from a series of trehalose glasses dild. with glycerol. A strong correlation with recently published protein stability data in the same series of glasses illustrates that the dynamics at Q ≥ 0.71 Å-1 and ω > 200 MHz are important to stabilization of horseradish peroxidase and yeast alc. dehydrogenase in these glasses. To the best of our knowledge, this is the first direct evidence that enzyme stability in a room temp. glass depends upon suppressing these short-length scale, high-frequency dynamics within the glass. We briefly discuss the coupling of protein motions to the local dynamics of the glass. Also, we show that the glass transition temp. (Tg) alone is not a good indicator for the protein stability in this series of glasses; the glass that confers the max. room-temp. stability does not have the highest Tg.
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    Soles, C. L.; Tsai, A. M.; Cicerone, M. T. Misbehaving Proteins: Protein (Mis)Folding, Aggregation, and Stability; Springer New York: New York, 2006; pp 193– 214,  DOI: 10.1007/978-0-387-36063-8_9 .
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    Cornicchi, E.; Onori, G.; Paciaroni, A. Picosecond-Time-Scale Fluctuations of Proteins in Glassy Matrices: The Role of Viscosity. Phys. Rev. Lett. 2005, 95, 158104,  DOI: 10.1103/PhysRevLett.95.158104
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    Picosecond-Time-Scale Fluctuations of Proteins in Glassy Matrices: The Role of Viscosity
    Cornicchi, Elena; Onori, Giuseppe; Paciaroni, Alessandro
    Physical Review Letters (2005), 95 (15), 158104/1-158104/4CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)
    Through elastic neutron scattering we investigated the fast dynamics of lysozyme in hydrated powder form or embedded in glycerol-water and glucose-water matrixes. We calcd. the relaxational contribution to the mean square displacements of protein hydrogen atoms. We found that the inverse of this quantity is linearly proportional to the logarithm of the viscosity of the solvent glassy matrix. This relationship suggests a close connection between the picosecond-time-scale dynamics of protein side chains and the solvent structural relaxation.
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    Wuttke, J.; Budwig, A.; Drochner, M.; Kämmerling, H.; Kayser, F.-J.; Kleines, H.; Ossovyi, V.; Pardo, L. C.; Prager, M.; Richter, D.; Schneider, G. J.; Schneider, H.; Staringer, S. SPHERES, Jülich’s high-flux neutron backscattering spectrometer at FRM II. Rev. Sci. Instrum. 2012, 83, 075109,  DOI: 10.1063/1.4732806
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    SPHERES, Juelich's high-flux neutron backscattering spectrometer at FRM II
    Wuttke, Joachim; Budwig, Alfred; Drochner, Matthias; Kaemmerling, Hans; Kayser, Franz-Joseph; Kleines, Harald; Ossovyi, Vladimir; Pardo, Luis Carlos; Prager, Michael; Richter, Dieter; Schneider, Gerald J.; Schneider, Harald; Staringer, Simon
    Review of Scientific Instruments (2012), 83 (7), 075109/1-075109/11CODEN: RSINAK; ISSN:0034-6748. (American Institute of Physics)
    SPHERES is a third-generation neutron backscattering spectrometer, located at the 20 MW German neutron source FRM II and operated by the Juelich Center for Neutron Science. It offers an energy resoln. (fwhm) better than 0.65 μeV, a dynamic range of ± 31 μeV, and a signal-to-noise ratio of up to 1750:1. (c) 2012 American Institute of Physics.
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    Zamponi, M.; Khaneft, M. SPHERES: Backscattering spectrometer. J. Large-Scale Res. Facil. 2015, 1, A30,  DOI: 10.17815/jlsrf-1-38
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    Natali, F.; Peters, J.; Russo, D.; Barbieri, S.; Chiapponi, C.; Cupane, A.; Deriu, A.; Di Bari, M. T.; Farhi, E.; Gerelli, Y.; Mariani, P.; Paciaroni, A.; Rivasseau, C.; Schiro, G.; Sonvico, F. IN13 Backscattering Spectrometer at ILL: Looking for Motions in Biological Macromolecules and Organisms. Neutron News 2008, 19, 14– 18,  DOI: 10.1080/10448630802474083
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    Protein dynamics: comparison of simulations with inelastic neutron scattering experiments
    Smith, J. C.
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    A review with many refs.
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    Yi, Z.; Miao, Y.; Baudry, J.; Jain, N.; Smith, J. C. Derivation of Mean-Square Displacements for Protein Dynamics from Elastic Incoherent Neutron Scattering. J. Phys. Chem. B 2012, 116, 5028– 5036,  DOI: 10.1021/jp2102868
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    Derivation of Mean-Square Displacements for Protein Dynamics from Elastic Incoherent Neutron Scattering
    Yi, Zheng; Miao, Yinglong; Baudry, Jerome; Jain, Nitin; Smith, Jeremy C.
    Journal of Physical Chemistry B (2012), 116 (16), 5028-5036CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
    The derivation of mean-square displacements from elastic incoherent neutron scattering (EINS) of proteins was examd., with the aid of expts. on camphor-bound cytochrome P450cam and complementary mol. dynamics simulations. A q4 correction to the elastic incoherent structure factor (q is the scattering vector) can be simply used to reliably est. from the expt. both the av. mean-square at. displacement, 〈Δr2〉 of the nonexchanged hydrogen atoms in the protein and its variance, σ2. The mol. dynamics simulation results are in broad agreement with the exptl. derived 〈Δr2〉 and σ2 derived from EINS on instruments at two different energy resolns., corresponding to dynamics on the ∼100 ps and ∼1 ns time scales. Significant dynamical heterogeneity is found to arise from methyl-group rotations. The easy-to-apply q4 correction extends the information extd. from elastic incoherent neutron scattering expts. and should be of wide applicability.
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    Parak, F.; Knapp, E. W. A consistent picture of protein dynamics. Proc. Natl. Acad. Sci. U. S. A. 1984, 81, 7088– 7092,  DOI: 10.1073/pnas.81.22.7088
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    A consistent picture of protein dynamics
    Parak, F.; Knapp, E. W.
    Proceedings of the National Academy of Sciences of the United States of America (1984), 81 (22), 7088-92CODEN: PNASA6; ISSN:0027-8424.
    Information about the protein dynamics of myoglobin obtained by x-ray and Moessbauer investigations is analyzed and compared with computer simulations. Computer simulations give correct amplitudes of mean-square displacements but fail in the description of the time dependence of motions. A proposed model describes protein dynamics at physiol. temps. as an overdamped diffusion-like motion in a restricted space. The fluctuations occur around the av. conformation detd. by x-ray structure anal. The gain in entropy drives the mol. into the transition state and, in this way, accounts for its flexibility.
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    Zeller, D.; Telling, M. T. F.; Zamponi, M.; García-Sakai, V.; Peters, J. Analysis of elastic incoherent neutron scattering data beyond the Gaussian approximation. J. Chem. Phys. 2018, 149, 234908,  DOI: 10.1063/1.5049938
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    Analysis of elastic incoherent neutron scattering data beyond the Gaussian approximation
    Zeller, D.; Telling, M. T. F.; Zamponi, M.; Garcia Sakai, V.; Peters, J.
    Journal of Chemical Physics (2018), 149 (23), 234908/1-234908/17CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
    This work addresses the use of the Gaussian approxn. as a common tool to ext. at. motions in proteins from elastic incoherent neutron scattering and whether improvements in data anal. and addnl. information can be obtained when going beyond that. We measured alpha-lactalbumin with different levels of hydration on three neutron backscattering spectrometers, to be able to resolve a wide temporal and spatial range for dynamics. We demonstrate that the Gaussian approxn. gives qual. similar results to models that include heterogeneity, if one respects a certain procedure to treat the intercept of the elastic intensities with the momentum transfer axis. However, the inclusion of motional heterogeneity provides better fits to the data. Our anal. suggests an approach of limited heterogeneity, where including only two kinds of motions appears sufficient to obtain more quant. results for the mean square displacement. Finally, we note that traditional backscattering spectrometers pose a limit on the lowest accessible momentum transfer. We therefore suggest that complementary information about the spatial evolution of the elastic intensity close to zero momentum transfer can be obtained using other neutron methods, in particular, neutron spin-echo together with polarization anal. (c) 2018 American Institute of Physics.
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    Frauenfelder, H.; Sligar, S. G.; Wolynes, P. G. The energy landscapes and motions of proteins. Science 1991, 254, 1598– 1603,  DOI: 10.1126/science.1749933
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    The energy landscapes and motions of proteins
    Frauenfelder, Hans; Sligar, Stephen G.; Wolynes, Peter G.
    Science (Washington, DC, United States) (1991), 254 (5038), 1598-603CODEN: SCIEAS; ISSN:0036-8075.
    Recent expts., advances in theory, and analogies to other complex systems such as glasses and spin glasses yield insight into protein dynamics. The basis of the understanding is the observation that the energy landscape is complex: proteins can assume a large no. of nearly isoenergetic conformations (conformational substates). The concepts that emerge from studies of the conformational substates and the motions between them permit a quant. discussion of one simple reaction, the binding of small ligands such as carbon monoxide to myoglobin.
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    Paciaroni, A.; Cinelli, S.; Cornicchi, E.; Francesco, A. D.; Onori, G. Fast fluctuations in protein powders: The role of hydration. Chem. Phys. Lett. 2005, 410, 400– 403,  DOI: 10.1016/j.cplett.2005.05.098
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    Fast fluctuations in protein powders: The role of hydration
    Paciaroni, Alessandro; Cinelli, Stefania; Cornicchi, Elena; De Francesco, Alessio; Onori, Giuseppe
    Chemical Physics Letters (2005), 410 (4-6), 400-403CODEN: CHPLBC; ISSN:0009-2614. (Elsevier B.V.)
    An elastic neutron scattering investigation of the mol. dynamics of hydrated lysozyme powders has been undertaken for different water contents, h (g water/g Lysozyme). The dry sample exhibits a harmonic behavior in the whole temp. range, while anharmonic motions arise on hydrated samples at a temp. T d. Both T d and the magnitude of the anharmonic motions are markedly hydration-dependent. On increasing water content, the crossing barrier entropy change increases, while the enthalpy change keeps const. The estd. av. rigidity of the protein structure decreases abruptly immediately below the onset of the enzymic activation at around 0.2h.
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    Schiró, G.; Natali, F.; Cupane, A. Physical Origin of Anharmonic Dynamics in Proteins: New Insights From Resolution-Dependent Neutron Scattering on Homomeric Polypeptides. Phys. Rev. Lett. 2012, 109, 128102,  DOI: 10.1103/PhysRevLett.109.128102
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    Physical origin of anharmonic dynamics in proteins: new insights from resolution-dependent neutron scattering on homomeric polypeptides
    Schiro, Giorgio; Natali, Francesca; Cupane, Antonio
    Physical Review Letters (2012), 109 (12), 128102/1-128102/5CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)
    Neutron scattering reveals a complex dynamics in polypeptide chains, with two main onsets of anharmonicity whose phys. origin and biol. role are still debated. In this study the dynamics of strategically selected homomeric polypeptides is investigated with elastic neutron scattering using different energy resolns. and compared with that of a real protein. Our data spotlight the dependence of anharmonic transition temps. and fluctuation amplitudes on energy resoln., which we quant. explain in terms of a two-site model for the protein-hydration water energy landscape. Exptl. data strongly suggest that the protein dynamical transition is not a mere resoln. effect but is due to a real phys. effect. Activation barriers and free energy values obtained for the protein dynamical transition allow us to make a connection with the two-well interaction potential of supercooled-confined water proposed to explain a low-d. → high-d. liq.-liq. transition.
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    Stoeckli, A.; Furrer, A.; Schoenenberger, C.; Meier, B.; Ernst, R.; Anderson, I. Dynamics of hydrogen bonds in carboxylic acids. Physica B+C 1986, 136, 161– 164,  DOI: 10.1016/S0378-4363(86)80045-6
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    Stoeckli, A.; Furrer, A.; Schoenenberger, C.; Meier, B. H.; Ernst, R. R.; Anderson, I.
    Physica B+C: Physics of Condensed Matter + Atomic, Molecular and Plasma Physics, Optics (Amsterdam) (1986), 136 (1-3), 161-4CODEN: PHBCDQ; ISSN:0378-4363.
    Double proton exchange in cryst. ring-deuterated terephthalic acid and cryst. acetylenedicarboxylic acid was studied by incoherent neutron scattering. The energy spectra above 100 K reveal quasielastic contributions which indicate that the disorder of the H-bonded dimeric units is dynamic involving a translational jump across the hydrogen bonds. The data were analyzed in terms of a two-site jump model.
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    Schiró, G. Anharmonic onsets in polypeptides revealed by neutron scattering: Experimental evidences and quantitative description of energy resolution dependence. Biophys. Chem. 2013, 180–181, 29– 36,  DOI: 10.1016/j.bpc.2013.05.006
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    Anharmonic onsets in polypeptides revealed by neutron scattering: Experimental evidences and quantitative description of energy resolution dependence
    Schiro, Giorgio
    Biophysical Chemistry (2013), 180-181 (), 29-36CODEN: BICIAZ; ISSN:0301-4622. (Elsevier B.V.)
    Neutron scattering measurements on protein powders reveal 2 deviations from harmonic dynamics at low temp., whose mol. origin, phys. nature, and biol. relevance are still matter of discussion. Here, the author presents a new exptl. and theor. approach to evidence the resoln. dependence of anharmonic onsets: the use of strategically selected homomeric polypeptides, such as polyglycine and polyalanine, allowed revealing the exact resoln. dependence; a 2-site energy landscape model, where resoln. effects are explicitly taken into account, was able to interpret quant. the exptl. data in terms of energy landscape parameters. The energetic description provided by this anal., together with recent exptl. evidences obtained on chem. and structurally different peptide systems, allowed one to connect the protein/water energy landscape structure with the 2-wells water interaction potential proposed to explain the low-d. → high-d. liq.-liq. transition obsd. in supercooled water.
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    Afroze, F.; Nies, E.; Berghmans, H. Phase transitions in the system poly(N-isopropylacrylamide)/water and swelling behaviour of the corresponding networks. J. Mol. Struct. 2000, 554, 55– 68,  DOI: 10.1016/S0022-2860(00)00559-7
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    Phase transitions in the system poly(N-isopropylacrylamide)/water and swelling behavior of the corresponding networks
    Afroze, F.; Nies, E.; Berghmans, H.
    Journal of Molecular Structure (2000), 554 (1), 55-68CODEN: JMOSB4; ISSN:0022-2860. (Elsevier Science B.V.)
    The phase behavior of linear poly(N-isopropylacrylamide) (PNIPA) and chem. cross-linked PNIPA in water has been detd. by calorimetric and optical techniques. Expts. for the linear polymer were conducted for three different molar masses and encompassed the whole accessible concn. range. In addn. the binodal of the 'monomer' N-(isopropyl)propionamide (NIPPA) was detd. by cloud point measurements. The phase behavior was analyzed in terms of the Flory-Huggins-Staverman theory amended with a strong concn. dependent interaction function. The anal. established that PNIPA/water is exemplar of Type II phase behavior, indicating that the system does not fit the classic Flory-Huggins scheme. Distinctive of Type II behavior is the existence of an off-zero liq.-liq. crit. compn. for a hypothetical polymer of infinite molar wt. and the absence of the classic FH Θ-crit. state. The Type II phase behavior of the linear polymer is also found in the network and is shown to be responsible for the discontinuous vol. response of the PNIPA gel. The peculiar swelling behavior is a direct consequence of the invariant interference of the swelling curve with the liq.-liq. miscibility gap. Finally, the dependence of the enthalpy of melting of water in the polymer soln. is not related to the formation of a strong complex between the polymer and the water mols. Instead, crystn. and melting of water are impeded by the vitrification of the aq. polymer soln.
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    Van Durme, K.; Van Assche, G.; Van Mele, B. Kinetics of Demixing and Remixing in Poly(N-isopropylacrylamide)/Water Studied by Modulated Temperature DSC. Macromolecules 2004, 37, 9596– 9605,  DOI: 10.1021/ma048472b
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    Kinetics of Demixing and Remixing in Poly(N-isopropylacrylamide)/Water Studied by Modulated Temperature DSC
    Van Durme, Kurt; Van Assche, Guy; Van Mele, Bruno
    Macromolecules (2004), 37 (25), 9596-9605CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
    The heat capacity signal from modulated temp. DSC can be used to measure the onset of phase sepn. in aq. poly(N-isopropylacrylamide) (PNIPAM) solns., showing a type II LCST (lower crit. soln. temp.) demixing behavior. Quasi-isothermal measurements through the phase transition show large excess contributions in the (apparent) heat capacity, caused by demixing and remixing heat effects on the time scale of the modulation. These excess contributions and their time-dependent evolution are useful to describe the kinetics of phase sepn. and to follow the related morphol. development. Partial vitrification of the polymer-rich phase slows down the remixing kinetics.
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    Buratti, E.; Tavagnacco, L.; Zanatta, M.; Chiessi, E.; Buoso, S.; Franco, S.; Ruzicka, B.; Angelini, R.; Orecchini, A.; Bertoldo, M.; Zaccarelli, E. The role of polymer structure on water confinement in poly(N-isopropylacrylamide) dispersions. J. Mol. Liq. 2022, 355, 118924,  DOI: 10.1016/j.molliq.2022.118924
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    The role of polymer structure on water confinement in poly(N-isopropylacrylamide) dispersions
    Buratti, Elena; Tavagnacco, Letizia; Zanatta, Marco; Chiessi, Ester; Buoso, Sara; Franco, Silvia; Ruzicka, Barbara; Angelini, Roberta; Orecchini, Andrea; Bertoldo, Monica; Zaccarelli, Emanuela
    Journal of Molecular Liquids (2022), 355 (), 118924CODEN: JMLIDT; ISSN:0167-7322. (Elsevier B.V.)
    Poly(N-isopropylacrylamide) (PNIPAM) is a synthetic polymer that is widely studied for its thermoresponsive character. However, recent works also reported evidence of a low temp. (protein-like) dynamical transition around 225 K in concd. PNIPAM suspensions, independently of the polymer architecture, i.e., both for linear chains and for microgels. In this work, we investigate water-polymer interactions by extensive differential scanning calorimetry (DSC) measurements of both systems, in order to understand the effect of the different topol. structures on the soln. behavior, in particular regarding crystn. and melting processes. In addn., we compare protiated and deuterated microgels, in both water and deuterated water. The DSC results are complemented by dynamic light scattering expts., which confirm that the selective isotopic substitution differently affects the soln. behavior. Our findings highlight the important role played by the polymer architecture on the soln. behavior: indeed, microgels turn out to be more efficient confining agents, able to avoid water crystn. in a wider concn. range with respect to linear chains. Altogether, the present data will be valuable to interpret future low-temp. investigations of PNIPAM dispersions, particularly by neutron scattering expts.
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    States of water in different hydrophilic polymers - DSC and FTIR studies
    Ping, Z. H.; Nguyen, Q. T.; Chen, S. M.; Zhou, J. Q.; Ding, Y. D.
    Polymer (2001), 42 (20), 8461-8467CODEN: POLMAG; ISSN:0032-3861. (Elsevier Science Ltd.)
    The structure of water mols. sorbed in different hydrophilic polymers was studied by DSC and FTIR. The obtained data shows that, first, the sorbed water mols. are directly bound to the hydrophilic site to form non-freezable water. Then, beyond a certain water content threshold, the sorbed water mols. become freezable, but with a m.p. lower than 0°C, due to their location in the second hydration layer. Bulk-like water which can be frozen at 0°C appears at higher water contents, and the two types of freezable water finally merge together at very high water contents. The av. no. of non-freezable water mols. per site depends on the chem. nature of the polar site: ca. 1 for a hydroxyl, and 4.2 for an amide group. For a polymer with carboxylate sites, it increases with the size of the alk. counter-ion of the site, due to the increasing ability of the carboxylate counter-ion pair to undergo dissocn.
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    Zhang, T.; Li, T.; Nies, E.; Berghmans, H.; Ge, L. Isothermal crystallization study on aqueous solution of poly(vinyl methyl ether) by DSC method. Polymer 2009, 50, 1206– 1213,  DOI: 10.1016/j.polymer.2008.12.038
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    Isothermal crystallization study on aqueous solution of poly(vinyl methyl ether) by DSC method
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    Polymer (2009), 50 (5), 1206-1213CODEN: POLMAG; ISSN:0032-3861. (Elsevier Ltd.)
    A study on the isothermal crystn. of water in aq. solns. of poly(vinyl Me ether) (PVME) was carried out by the differential scanning calorimetry (DSC). The influence of PVME concn. (49.5, 44.5 and 39.5 v%) and the crystn. temp. (T c) on crystn. rate G, crystn. enthalpy (ΔH c) and melting enthalpy (ΔH m) was investigated. Avrami equation cannot be used to describe the crystn. process of water in aq. PVME soln. Within the measured temp. range, the crystn. rate G increases with the crystn. temp. T c and with the decreasing PVME content. The crystn. enthalpy ΔH c linearly increases with the degree of supercooling. The influence of T c on the ΔH c becomes more marked with increasing PVME concn. For 49.5 and 44.5 v% PVME solns., the amt. of water arrested in soln. during the isothermal crystn. and the final concn. of PVME-rich phase increase linearly with the T c, whereas for 39.5 v% PVME soln., these two values almost do not change with T c. The amt. of frozen water in the subsequent cold crystn. is approx. proportional to the initial T c. The approx. const. ΔH m for a given concn. at the different initial isothermal crystn. temps. suggests that the total amt. of ice from the first isothermal crystn. and the second cold crystn. is same. The quant. relation of the amt. of frozen water in the cold crystn. and the initial T c demonstrates that PVME/water complexes are thermodynamically unstable.
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    Tanaka, M.; Motomura, T.; Ishii, N.; Shimura, K.; Onishi, M.; Mochizuki, A.; Hatakeyama, T. Cold crystallization of water in hydrated poly(2-methoxyethyl acrylate) (PMEA). Polym. Int. 2000, 49, 1709– 1713,  DOI: 10.1002/1097-0126(200012)49:12<1709::AID-PI601>3.0.CO;2-L
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    Cold crystallization of water in hydrated poly(2-methoxyethyl acrylate) (PMEA)
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    Polymer International (2000), 49 (12), 1709-1713CODEN: PLYIEI; ISSN:0959-8103. (John Wiley & Sons Ltd.)
    The structure of water assocd. with poly(2-methoxyethyl acrylate) (PMEA), which is known to exhibit excellent blood compatibility, has been investigated using DSC. The total equil. water content (EWC) of PMEA was 9.0wt%. Water in the PMEA could be classified into three types: non-freezing, freezing-bound and free water. Cold crystn. of water was clearly obsd. at about -42°C on heating when the water content was more than 3.0wt%. Cold crystn. is attributed to the phase transition from the amorphous ice to the crystal ice in PMEA. The relative proportions of freezing-bound water at the EWC is 48% of all the water in hydrated PMEA.
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    Guan, L.; Xu, H.; Huang, D. The investigation on states of water in different hydrophilic polymers by DSC and FTIR. Journal of Polymer Research 2011, 18, 681– 689,  DOI: 10.1007/s10965-010-9464-7
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    The investigation on states of water in different hydrophilic polymers by DSC and FTIR
    Guan, Lan; Xu, Hongyan; Huang, Dinghai
    Journal of Polymer Research (2011), 18 (4), 681-689CODEN: JPOREP; ISSN:1022-9760. (Springer)
    The interaction between polymers and water in four hydrophilic polymer aq. solns. were investigated by DSC and FTIR. DSC result shows that the different hydrophilic polymer/water mixts. have various water calorimetric behaviors in the melting temp. range of freezable bound water as well as free water. The melting temp. of freezable water and the amt. of non-freezable water in the mixts. vary with the change of chem. structure of polymers. The m.p. of the freezable bound water doesn't change with the water content, revealing that water bound weakly to polymer chains can form a stable cryst. structure at high water content. For the three hydrophilic polymer/water mixts. with C=O group, the wt. ratio of non-freezable water to polymers is const., but varies with polymer chem. structures. The FTIR spectra confirmed the formation of the hydrogen bonds and it was found that there exist different states of water based on various strengths of hydrogen bonds. The OH stretching bands indicated the fraction of strongly bound water decreases with increasing water content. It was concluded that at least in hydrophilic polymer aq. solns. with polar sites in polymer chains, the formation of non-freezable water is ascribed to the hydrogen bonds between hydrophilic polymers and water mols. Different strengths of hydrogen bonds can affect the thermal behaviors of water in the hydrophilic polymer/water mixts.
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    Guo, Y.; Sun, B.; Wu, P. Phase Separation of Poly(vinyl methyl ether) Aqueous Solution: A Near-Infrared Spectroscopic Study. Langmuir 2008, 24, 5521– 5526,  DOI: 10.1021/la7038398
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    Phase Separation of Poly(vinyl methyl ether) Aqueous Solution: A Near-Infrared Spectroscopic Study
    Guo, Yilu; Sun, Bingjie; Wu, Peiyi
    Langmuir (2008), 24 (10), 5521-5526CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    The thermosensitive phase sepn. of poly(vinyl Me ether) (PVME) aq. solns. was studied using near-IR spectroscopy in combination with two-dimensional correlation anal., and a two-step phase sepn. mechanism during gradual heating was established. Two-dimensional near-IR (2D NIR) anal. results indicate that during this two-step process the dehydration of CH2 groups occurs earlier than that of CH3 groups. This result suggests that it is the change of the hydrophobic hydrocarbon chain conformation induced by heating that indirectly leads to the dehydration of the hydrophilic ether oxygen side groups.
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    Nickels, J. D.; O’Neill, H.; Hong, L.; Tyagi, M.; Ehlers, G.; Weiss, K. L.; Zhang, Q.; Yi, Z.; Mamontov, E.; Smith, J. C.; Sokolov, A. P. Dynamics of Protein and its Hydration Water: Neutron Scattering Studies on Fully Deuterated GFP. Biophys. J. 2012, 103, 1566– 1575,  DOI: 10.1016/j.bpj.2012.08.046
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    Dynamics of Protein and its Hydration Water: Neutron Scattering Studies on Fully Deuterated GFP
    Nickels, Jonathan D.; O'Neill, Hugh; Hong, Liang; Tyagi, Madhusudan; Ehlers, Georg; Weiss, Kevin L.; Zhang, Qiu; Yi, Zheng; Mamontov, Eugene; Smith, Jeremy C.; Sokolov, Alexei P.
    Biophysical Journal (2012), 103 (7), 1566-1575CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    We present a detailed anal. of the picosecond-to-nanosecond motions of green fluorescent protein (GFP) and its hydration water using neutron scattering spectroscopy and hydrogen/deuterium contrast. The anal. reveals that hydration water suppresses protein motions at lower temps. (<∼200 K), and facilitates protein dynamics at high temps. Exptl. data demonstrate that the hydration water is harmonic at temps. <∼180-190 K and is not affected by the proteins' Me group rotations. The dynamics of the hydration water exhibits changes at ∼180-190 K that we ascribe to the glass transition in the hydrated protein. Our results confirm significant differences in the dynamics of protein and its hydration water at high temps.: on the picosecond-to-nanosecond timescale, the hydration water exhibits diffusive dynamics, while the protein motions are localized to <∼3 Å. The diffusion of the GFP hydration water is similar to the behavior of hydration water previously obsd. for other proteins. Comparison with other globular proteins (e.g., lysozyme) reveals that on the timescale of 1 ns and at equiv. hydration level, GFP dynamics (mean-square displacements and quasielastic intensity) are of much smaller amplitude. Moreover, the suppression of the protein dynamics by the hydration water at low temps. appears to be stronger in GFP than in other globular proteins. We ascribe this observation to the barrellike structure of GFP.
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    Sebastiani, F.; Longo, M.; Orecchini, A.; Comez, L.; De Francesco, A.; Muthmann, M.; Teixeira, S. C. M.; Petrillo, C.; Sacchetti, F.; Paciaroni, A. Hydration-dependent dynamics of human telomeric oligonucleotides in the picosecond timescale: A neutron scattering study. J. Chem. Phys. 2015, 143, 015102,  DOI: 10.1063/1.4923213
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    Hydration-dependent dynamics of human telomeric oligonucleotides in the picosecond timescale: A neutron scattering study
    Sebastiani, F.; Longo, M.; Orecchini, A.; Comez, L.; De Francesco, A.; Muthmann, M.; Teixeira, S. C. M.; Petrillo, C.; Sacchetti, F.; Paciaroni, A.
    Journal of Chemical Physics (2015), 143 (1), 015102/1-015102/8CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
    The dynamics of the human oligonucleotide, AG3(T2AG3)3, were investigated by incoherent neutron scattering in the sub-nanosecond timescale. A hydration-dependent dynamical activation of thermal fluctuations in weakly hydrated samples was found, similar to that of protein powders. The amplitudes of such thermal fluctuations were evaluated in 2 different exchanged wave-vector ranges, so as to single out the different contributions from intra- and inter-nucleotide dynamics. The activation energy was calcd. from the temp.-dependent characteristic times of the corresponding dynamical processes. The trends of both amplitudes and activation energies support a picture where oligonucleotides possess a larger conformational flexibility than long DNA sequences. This addnl. flexibility, which likely results from a significant relative chain-end contribution to the av. chain dynamics, could be related to the strong structural polymorphism of the investigated oligonucleotides. (c) 2015 American Institute of Physics.
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    Russo, D.; Gonzalez, M. A.; Pellegrini, E.; Combet, J.; Ollivier, J.; Teixeira, J. Evidence of Dynamical Constraints Imposed by Water Organization around a Bio–Hydrophobic Interface. J. Phys. Chem. B 2013, 117, 2829– 2836,  DOI: 10.1021/jp3094885
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    Evidence of dynamical constraints imposed by water organization around a bio-hydrophobic interface
    Russo, Daniela; Gonzalez, Miguel Angel; Pellegrini, Eric; Combet, J.; Ollivier, J.; Teixeira, Jose
    Journal of Physical Chemistry B (2013), 117 (10), 2829-2836CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
    Mol. dynamics simulations and elastic neutron scattering expts. were used to highlight how the structural organization of hydration water is able in some cases to locally constrain at. movements at biol. interfaces. Using fully hydrated small peptides as models of protein interfaces, the authors show that the length of the side-chains and the hydrophilic backbone have specific signatures. The dynamics of the side-chain, which is part of biomols., play not only a crucial role in the whole flexibility as compared to the backbone, but also modify the values of transition temps. The anal. of the activation energies of Me group dynamics suggests that the interaction between a hydrophobic side-chain and the surrounding water plays an important role in the whole flexibility as well. The authors suggest that the progressive water cluster organization, around hydrophobic interfaces increases the activation energy and that a plateau regime is reached only when an extended H-bond network is established. The cluster size corresponds to a single layer of water mols.
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    Hong, L.; Smolin, N.; Lindner, B.; Sokolov, A. P.; Smith, J. C. Three Classes of Motion in the Dynamic Neutron-Scattering Susceptibility of a Globular Protein. Phys. Rev. Lett. 2011, 107, 148102,  DOI: 10.1103/PhysRevLett.107.148102
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    Three Classes of Motion in the Dynamic Neutron-Scattering Susceptibility of a Globular Protein
    Hong, Liang; Smolin, Nikolai; Lindner, Benjamin; Sokolov, Alexei P.; Smith, Jeremy C.
    Physical Review Letters (2011), 107 (14), 148102/1-148102/4CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)
    A simplified description of the 295 K dynamics of a globular protein over a wide frequency range (1-1000 GHz) is obtained by combining neutron scattering of lysozyme with mol. dynamics (MD) simulation. The mol. dynamics simulation agrees quant. with expt. for both the protein and the hydration water and shows that whereas the hydration water mols. subdiffuse, the protein atoms undergo confined motion decomposable into three distinct classes: localized diffusion, Me group rotations, and jumps. Each of the three classes gives rise to a characteristic neutron susceptibility signal.
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    Schiró, G.; Caronna, C.; Natali, F.; Cupane, A. Direct Evidence of the Amino Acid Side Chain and Backbone Contributions to Protein Anharmonicity. J. Am. Chem. Soc. 2010, 132, 1371– 1376,  DOI: 10.1021/ja908611p
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    Direct Evidence of the Amino Acid Side Chain and Backbone Contributions to Protein Anharmonicity
    Schiro, Giorgio; Caronna, Chiara; Natali, Francesca; Cupane, Antonio
    Journal of the American Chemical Society (2010), 132 (4), 1371-1376CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Elastic incoherent neutron scattering has been used to study the temp. dependence of the mean-square displacements of nonexchangeable hydrogen atoms in powders of a series of homomeric polypeptides (polyglycine, polyalanine, polyphenylalanine, and polyisoleucine) in comparison with myoglobin at the same hydration level (h = 0.2). The aim of the work was to measure the dynamic behavior of different amino acid residues sep. and assess the contribution of each type of side chain to the anharmonic dynamics of proteins. The results provide direct exptl. evidence that the first anharmonic activation, at ∼150 K, is largely due to Me group rotations entering the time window of the spectrometer used; however, contributions on the order of 10-20% from the motions of other groups (e.g., the phenolic ring and the methylene groups) are present. Our data also indicate that the dynamical transition occurring at ∼230 K can be attributed, at least at the hydration level investigated, mainly to motions involving backbone fluctuations.
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    Schiró, G.; Caronna, C.; Natali, F.; Cupane, A. Molecular origin and hydration dependence of protein anharmonicity: An elastic neutron scattering study. Phys. Chem. Chem. Phys. 2010, 12, 10215– 10220,  DOI: 10.1039/c003482g
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    Molecular origin and hydration dependence of protein anharmonicity: An elastic neutron scattering study
    Schiro, Giorgio; Caronna, Chiara; Natali, Francesca; Cupane, Antonio
    Physical Chemistry Chemical Physics (2010), 12 (35), 10215-10220CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)
    Two main onsets of anharmonicity are present in protein dynamics. Neutron scattering on protein hydrated powders revealed a first onset at about 150 K and a second one at about 230 K (the so called dynamical transition). In order to assess the mol. origin of protein anharmonicity, we study different homomeric polypeptides by incoherent elastic neutron scattering, thus disentangling the contribution of different mol. groups in proteins. We show that Me group rotations are the main contributors to the low temp. onset. Concerning the dynamical transition, we show that it also occurs in absence of side chains; however, the presence and mobility of side chains substantially increases the fluctuations amplitude without influencing the transition temp. We also investigate the role of hydration on the anharmonic contributions. Our study shows that Me group rotations are unaffected by hydration and confirms that the dynamical transition is suppressed in dry samples. In hydrated samples, while the pure backbone contribution does not depend on the hydration h at h ≥ 0.2, in the presence of side chains the anharmonic fluctuations involved in the dynamical transition are enhanced by increasing the water content.
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    Roh, J. H.; Novikov, V. N.; Gregory, R. B.; Curtis, J. E.; Chowdhuri, Z.; Sokolov, A. P. Onsets of Anharmonicity in Protein Dynamics. Phys. Rev. Lett. 2005, 95, 038101,  DOI: 10.1103/PhysRevLett.95.038101
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    Physical Review Letters (2005), 95 (3), 038101/1-038101/4CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)
    Two onsets of anharmonicity are obsd. in the dynamics of the protein lysozyme. One at T∼100 K appears in all samples regardless of hydration level and is consistent with Me group rotation. The second, the well-known dynamical transition at T∼200-230 K, is only obsd. at a hydration level h greater than ∼0.2 and is ascribed to the activation of an addnl. relaxation process. Its variation with hydration correlates well with variations of catalytic activity suggesting that the relaxation process is directly related to the activation of modes required for protein function.
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    Frick, B.; Fetters, L. J. Methyl Group Dynamics in Glassy Polyisoprene: A Neutron Backscattering Investigation. Macromolecules 1994, 27, 974– 980,  DOI: 10.1021/ma00082a014
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    Methyl Group Dynamics in Glassy Polyisoprene: A Neutron Backscattering Investigation
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    Macromolecules (1994), 27 (4), 974-80CODEN: MAMOBX; ISSN:0024-9297.
    Energy-resolved, elastic neutron backscattering was employed to investigate the Me group dynamics in polyisoprene between T = 2 K and room temp. The use of partially deuterated samples and a fully protonated sample allowed the sepn. of the dynamics arising from the Me group and the backbone. An Arrhenius-like increase of the Me group rotational correlation time τ = τ0 exp(Eact/kT), with Eact/k = 1550 K ∼ 12 kJ/mol and Γ0 ∼ 1/τ0 = 23.5 meV (τ0 ∼ 1.76 × 10-13 s) describes the mid-position of the first elastic intensity decrease but not its breadth. A 3-fold jump model with a broad Gaussian distribution of activation energies (dE/E ∼ 25%) around 1500 K can account for the obsd. temp. decrease. Inconstancies in the Q-dependence might be due to disorder effects. The torsional mode of the Me group rotation is directly obsd. at Γ0 = 23.5 meV by time-of-flight. Near the glass transition temp. a further decrease of the elastic scattering is obsd. due to the onset of fast dynamics of the backbone in the picosecond range.
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    Rasmussen, D. H.; MacKenzie, A. P. Glass transition in amorphous water. Application of the measurements to problems arising in cryobiology. J. Phys. Chem. 1971, 75, 967– 973,  DOI: 10.1021/j100677a022
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    Rasmussen, Don H.; MacKenzie, Alan P.
    Journal of Physical Chemistry (1971), 75 (7), 967-73CODEN: JPCHAX; ISSN:0022-3654.
    Values for the glass transition temp., Tg, of aq. solns. of glycerol, ethylene glycol, and MeOH, measured by DTA, were extrapolated to obtain values for Tg in amorphous water. For a heating rate of 5 deg min-1, Tg for amorphous water is obsd. at -137 ± 1°. The results were correlated with Jenckel's expression for the concn. dependence of Tg in binary solns. A kinetic anal. after McMillan led to "kinetic" parameters for the glass transition that were linear functions of wt. fraction and yielded a time-temp. dependence for Tg in amorphous water correlating well with previously published values.
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    Bohon, R.; Conway, W. DTA studies on the glycerol─water system. Thermochim. Acta 1972, 4, 321– 341,  DOI: 10.1016/0040-6031(72)87016-3
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    Thermochimica Acta (1972), 4 (3), 321-41CODEN: THACAS; ISSN:0040-6031.
    A modified duPont 900 DTA cell was used to study the complex phase transitions in the glycerol-H2O system at relatively rapid rates of heating and cooling and under carefully monitored thermal history conditions. This system is of particular interest in the cryoprotection of red blood cells. The separation of crystals and a const.-compn. amorphous phase from H2O-rich systems tends to distort the glass transition (Tg) vs. compn. curves unless special precautions are exercised. Extrapolation of Tg to zero glycerol content gave a hypothetical 2nd-order transition in "amorphous" ice of -123 ± 1° at a heating rate of 30°/min. The glycerol-H2O system may exhibit the phenomena of vitreous polymorphs wherein 2 distinct amorphous phases coexist in metastable equil. in the solid state. The obsd. 2nd-order transition in 1 of these phases occurred near, and in some cases slightly above, Tg for pure glycerol. Limited low temp. x-ray diffraction studies on 43.7% glycerol showed the existence of 2 crystalline forms, 1 of which could only be formed by rapid cooling from the liq. phase. Neither form gave diffraction patterns corresponding to known forms of ice.
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    Hatakeyama, H.; Yoshida, H.; Nakano, J. Studies on the isothermal crystallization of D-glucose and cellulose oligosaccharides by differential scanning calorimetry. Carbohydr. Res. 1976, 47, 203– 211,  DOI: 10.1016/S0008-6215(00)84185-3
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    Studies on the isothermal crystallization of D-glucose and cellulose oligosaccharides by differential scanning calorimetry
    Hatakeyama, Hyoe; Yoshida, Hiroshisa; Nakano, Junzo
    Carbohydrate Research (1976), 47 (2), 203-11CODEN: CRBRAT; ISSN:0008-6215.
    Isothermal crystn. from the glassy state of D-glucose and cellulose oligosaccharides (e.g., cellobiose, cellotriose, and cellotetraose) was studied by differential scanning calorimetry. The crystn. of amorphous D-glucose and oligosaccharides was very difficult in the absence of traces of water. Amorphous cellobiose and cellotetraose crystd. far more rapidly than amorphous D-glucose and cellotriose. The activation energy for the crystn. of cellobiose and cellotetraose was ∼10-12 kJ-mole-1, while that for D-glucose and cellotriose was ∼1-2 kJ-mole-1. An odd-even effect seemed to be assocd. with the crystn. process of these saccharides.
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    Noel, T. R.; Parker, R.; Ring, S. G. A comparative study of the dielectric relaxation behaviour of glucose, maltose, and their mixtures with water in the liquid and glassy states. Carbohydr. Res. 1996, 282, 193– 206,  DOI: 10.1016/0008-6215(95)00388-6
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    A comparative study of the dielectric relaxation behavior of glucose, maltose, and their mixtures with water in the liquid and glassy states
    Noel, Timothy R.; Parker, Roger; Ring, Stephen G.
    Carbohydrate Research (1996), 282 (2), 193-206CODEN: CRBRAT; ISSN:0008-6215. (Elsevier)
    The dielec. relaxation behavior of glucose, maltose, and their mixts. with water up to a concn. of 12.0 and 23.0% wt./wt., resp., were examd. in the frequency range 102 to 105 Hz. A primary relaxation was obsd. at temps. above the glass transition temp., Tg, and a secondary relaxation at sub-Tg temps. The addn. of water shifted the primary relaxations to lower temps. For the glucose mixts., water increased the strength of the secondary relaxation and resulted in a merging of the primary and secondary relaxations. The increase in strength of the secondary relaxation was much more marked for the maltose-water mixts. and, in this case, the relaxations remained sep. over the range of frequency and water contents studied. For the maltose-water mixts., the dependence of the strength of the secondary relaxation on the water content was bilinear with a change in gradient at ∼10.0% wt./wt. water. The sub-Tg relaxations were thought to arise from motions of pendant hydroxymethyl groups attached to the hexose rings and from the reorientation of water mols. The difference in the secondary relaxation behavior of glucose and maltose indicates that structural factors, in addn. to the presence of hydroxymethyl groups, are also important.
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    Shpigelman, A.; Portnaya, I.; Ramon, O.; Livney, Y. D. Saccharide-structure effects on poly N-isopropylacrylamide phase transition in aqueous media; Reflections on protein stability. J. Polym. Sci., Part B: Polym. Phys. 2008, 46, 2307– 2318,  DOI: 10.1002/polb.21562
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    Saccharide-structure effects on poly N-isopropylacrylamide phase transition in aqueous media; reflections on protein stability
    Shpigelman, Avi; Portnaya, Irina; Ramon, Ory; Livney, Yoav D.
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    Protein stability in aq. solns. is important in numerous fields, particularly biotechnol. and food-science. To shed new light on the protective effect of carbohydrates on proteins, we studied saccharide-structure effects in aq. solns. on the coil-to-globule transition occurring at the lower crit. soln. temp. (LCST) of poly-N-isopropylacrylamide (PNIPA), an isomer of polyleucine, as a simple model representing certain key behaviors of proteins (e.g., denaturation/renaturation). We systematically selected sugars and polyols to relate structural and phys. characteristics of these carbohydrates to their effect on PNIPA solns. Using isothermal titrn.-microcalorimetry, we showed that no significant binding of saccharides to the polymer occurs. Using micro-DSC, we studied the decreasing polymer LCST temp. with rising carbohydrate concn. Beyond the expected observation that steric exclusion is important, we obsd. previously-unreported significant differences among the effects of isomeric aldohexoses and also among the effects of isomeric diglucoses on PNIPA LCST. We found good correlation between the sugar hydration no. and its effect on LCST. We conclude that the larger and denser the hydrated cluster a carbohydrate forms, the worse a cosolvent is for the polymer, and the stronger it's lowering effect of the coil-to-globule transition. Such favoring of the compact globule state provides a protective effect against denaturation of globular proteins.
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    Corezzi, S.; Bracco, B.; Sassi, P.; Paolantoni, M.; Comez, L. Protein Hydration in a Bioprotecting Mixture. Life 2021, 11, 995,  DOI: 10.3390/life11100995
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    Protein Hydration in a Bioprotecting Mixture
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    Life (Basel, Switzerland) (2021), 11 (10), 995CODEN: LBSIB7; ISSN:2075-1729. (MDPI AG)
    We combined broad-band depolarized light scattering and IR spectroscopies to study the properties of hydration water in a lysozyme-trehalose aq. soln., where trehalose is present above the concn. threshold (30% in wt.) relevant for biopreservation. The joint use of the two different techniques, which were sensitive to inter-and intra-mol. degrees of freedom, shed new light on the mol. mechanism underlying the interaction between the three species in the mixt. Thanks to the comparison with the binary soln. cases, we were able to show that, under the investigated conditions, the protein, through preferential hydration, remains strongly hydrated even in the ternary mixt. This supported the water entrapment scenario, for which a certain amt. of water between protein and sugar protects the biomol. from damage caused by external agents.
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    Shimizu, S.; Smith, D. J. Preferential hydration and the exclusion of cosolvents from protein surfaces. J. Chem. Phys. 2004, 121, 1148– 1154,  DOI: 10.1063/1.1759615
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    Protein stability is enhanced by the addn. of osmolytes, such as sugars and polyols and inert crowders, such as polyethylene glycols. This stability enhancement has been quantified by the preferential hydration parameter which can be detd. by expts. To understand the mechanism of protein stability enhancement, we present a statistical mech. anal. of the preferential hydration parameter based upon Kirkwood-Buff theory. Previously, the preferential hydration parameter was interpreted in terms of the no. of hydration waters, as well as the cosolvent exclusion vol. It was not clear how accurate these interpretations were, nor what the relationship is between the two. By using the Kirkwood-Buff theory and exptl. data, we conclude that the contribution from the cosolvent exclusion dominantly dets. the preferential hydration parameters for crowders. For osmolytes, although the cosolvent exclusion largely dets. the preferential hydration parameters, the contribution from hydration may not be negligible.
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  1. Neng Hu, Di Gao, Feihong Song, Chuanqi Yang, Jianqi Zhang, Peter Müller-Buschbaum, Qi Zhong. Effect of Embedded g-C3N4 Nanosheets on the Hydration and Thermal Response Behavior of Cross-Linked Thermoresponsive Copolymer Films. Langmuir 2024, 40 (28) , 14663-14673. https://doi.org/10.1021/acs.langmuir.4c01630
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  • Abstract

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    Scheme 1

    Scheme 1. Chemical Formula of the NIPAM Repeating Unita
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    aThe C atoms of the amide (hydrophilic) and isopropyl (hydrophobic) groups in the side chain are highlighted in blue and green, respectively. The exchangeable H atom in the amide group is highlighted in red.

    Figure 1

    Figure 1. Logarithm of the EINS intensities of PNIPAM:d-glyc 1h as a function of Q2 at selected temperatures. Experimental data are reported together with the double-well fitting functions (solid red lines). For comparison, corresponding Gaussian fitting functions (dashed green lines) are also reported.

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    Figure 2

    Figure 2. Logarithm of the integrated EINS intensities versus T for (a) PNIPAM, (b) PNIPAM:d-glyc, and (c) PNIPAM:d-glu. Data are rescaled by their value at T = 153 K. Gray bars and black straight lines are guides for the eye, helping to visualize the slope changes at Td occurring in the 1h sample of each series. Inset in (a): comparison between the of PNIPAM 1h collected on two different spectrometers.

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    Figure 3

    Figure 3. ⟨Δu2⟩tot of pure PNIPAM. For easier comparison of their slope, data were corrected for their value at 153 K (unsubtracted data are shown in the insets). Red lines result from the linear fit of ⟨Δu2⟩tot for the 1h sample. Arrows indicate the slope change at Td.

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    Figure 4

    Figure 4. ⟨Δu2⟩tot of PNIPAM:d-glyc at τR = 150 (a) and 2000 ps (b). MSDs are corrected for their value at T = 153 K. Solid lines and colored bars help to visualize for each sample the slope changes at Td. Further slope changes at Tin are indicated by arrows. In the inset, slope changes at Tg are highlighted.

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    Figure 5

    Figure 5. ⟨Δu2⟩tot of PNIPAM:d-glu at τR = 150 (a) and 2000 ps (b). MSDs are corrected for their value at T = 153 K. Solid lines and colored bars help to visualize for each sample the slope changes at Td. Further slope changes at Tin are indicated by arrows. In the insets, the slope changes at Tg are highlighted.

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    Figure 6

    Figure 6. Dynamical transition temperatures Td of (a) PNIPAM:d-glyc and (b) PNIPAM:d-glu as a function of h. Dashed red lines indicate the value of Td in PNIPAM 1h (without cosolvents).

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    Figure 7

    Figure 7. MSDs of PNIPAM, PNIPAM:d-glyc, and PNIPAM:d-glu in the high-hydration regime (1h). MSDs are corrected for the corresponding value at T = 153 K. For each sample, the corresponding values of Td, Tin, and Tm are indicated by arrows. Solid lines are obtained from the linear fit of the data in the temperature region below Td (1), above Td and below Tin (2), and above Tm (3).

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    Figure 8

    Figure 8. Pseudoforce constant k at τR = (a) 150 and (b) 2000 ps calculated for PNIPAM samples at 1h. Values of k are obtained from the MSDs slope in the temperature region (1) below Td (red), (2) between Td and Tin (blue), and (3) above Tm (yellow).

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    Figure 9

    Figure 9. MSDs of PNIPAM, PNIPAM:d-glyc, and PNIPAM:d-glu at the same hydration degree (0h). For each binary sample, the corresponding values of Td and Tg are indicated by arrows. Solid lines are obtained from the linear fit of the MSDs in the temperature range between 153 and 313 K, 153 and 203 K, 153 and 303 K, respectively, for PNIPAM, PNIPAM:d-glyc, and PNIPAM:d-glu at τR = 150 ps. All MSDs were fitted in the same temperature range between 153 and 209 K at τR = 2000 ps.

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    Figure 10

    Figure 10. Low-temperature pseudoforce constant k at τR = 150 (empty circles) and 2000 ps (solid squares) calculated for dry PNIPAM (0h) compared with values calculated for PNIPAM embedded in pure water (PNIPAM 1h), pure glycerol (PNIPAM:d-glyc 0h), and pure glucose (PNIPAM:d-glu 0h).

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  • References

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      Comparison based on statistical thermodynamics between globule-to-coil transition of poly(N-isopropylacrylamide) and cold denaturation of a protein
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      Journal of Molecular Liquids (2020), 317 (), 114129CODEN: JMLIDT; ISSN:0167-7322. (Elsevier B.V.)
      When the temp. T becomes sufficiently low, poly(N-isopropylacrylamide) (PNIPAM) and a protein, resp., cause the globule-to-coil transition and the cold denaturation (i.e., transitions to states comprising more extended structures). It is exptl. known for PNIPAM that the coil state is sol. in water but the globule state is insol. By contrast, both of the cold-denatured and native states of a protein are sol. Using our recently developed statistical-mech. theory combined with mol. models for water, we show that the two structural transitions share phys. the same mechanism but still the difference between PNIPAM and a protein in terms of the solubilities of the two states can be reproduced. The solute hydration can be decompd. into the two processes: the creation of a cavity matching the solute structure at the at. level in water (process 1: hydrophobic hydration); and the incorporation of solute-water van der Waals interaction potential followed by that of solute-water electrostatic interaction potential (process 2). The hydration free energies, energies, and entropies in processes 1 and 2 are denoted by μH,1 > 0 and μH,2 < 0, εVH,1 < 0 and εVH,2 < 0, and SVH,1 < 0 and SVH,2 < 0, resp. We find that the excluded-vol. (EV) terms in εVH,1 and SVH,1 are strongly dependent on T, whereas not only the sum of the water-accessible surface terms in εVH,1 and SVH,1 but also εVH,2 and SVH,2 remain essentially const. against a change in T. The EV term of SVH,1 becomes significantly smaller at low T, which is interpretable as the weakening of the hydrophobic effect and the trigger of the two structural transitions. The changes in structure and properties of water near PNIPAM or a protein upon the transition to a state comprising more extended structures are unimportant. Though μH,1 is a largely increasing function of T, |μH,2| is only very weakly dependent on T. μH,1/|μH,2| for PNIPAM is much larger than that for a protein, which is attributable to the lower electrostatic affinity of PNIPAM for water. As a consequence, μH(Coil) < 0 at low T but μH(Globule) » 0 at high T for PNIPAM but μH(Denatured) « 0 at low T and μH(Native) « 0 at high T for a protein (μH = μH,1 + μH,2).
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      Inoue, M.; Hayashi, T.; Hikiri, S.; Ikeguchi, M.; Kinoshita, M. Mechanism of globule-to-coil transition of poly(N-isopropylacrylamide) in water: Relevance to cold denaturation of a protein. J. Mol. Liq. 2019, 292, 111374,  DOI: 10.1016/j.molliq.2019.111374
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      Journal of Molecular Liquids (2019), 292 (), 111374pp.CODEN: JMLIDT; ISSN:0167-7322. (Elsevier B.V.)
      In water, poly(N-isopropylacrylamide) (PNIPAM) is in a sol. coil state below the lower crit. sol. temp. (LCST) but in an insol. globule state above LCST. Namely, as the temp. decreases, PNIPAM exhibits a globule-to-coil transition at LCST∼305 K. We generate structural ensembles of coil and globule states by all-atom mol. dynamics simulations conducted at 273 and 323 K, resp. We then calc. a variety of energetic and entropic components of thermodn. quantities of the two states at the two temps. using our recently developed, accurate statistical-mech. method for solute hydration where mol. models are employed for water and the PNIPAM structure is taken into account at the at. level. We identify the phys. factors driving or opposing the transition and evaluate their relative magnitudes and temp. dependences. The presence of PNIPAM generates an excluded vol. (EV) which is inaccessible to the centers of water mols. in the entire system. The presence of a water mol. also generates an EV for the other water mols. with the result that all of the water mols. are entropically correlated, causing water crowding. The globule state, where the EV is smaller and water crowding is less significant, is more favored in terms of the translational, configurational entropy of water. This effect always opposes the globule-to-coil transition. At low temps., however, this effect becomes significantly weaker, yielding to the factors driving it. The mechanism of the transition is phys. the same as that of cold denaturation of a protein.
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      The coil-to-globule conformational transition of poly(N-isopropylacrylamide) in aq. solns. occurred at ∼32° as detected by optical measurements. This transition temp. was unaffected by the polymer mol. wt. ((5-840) × 104) or concn. (0.01-1 wt.%). The heat of transition was of the order 10 cal/g polymer. The conformational transitions of poly(N-isopropylmethacrylamide) and methylcellulose in aq. solns. shown for comparison.
    12. 12
      Rosi, B. P.; Tavagnacco, L.; Comez, L.; Sassi, P.; Ricci, M.; Buratti, E.; Bertoldo, M.; Petrillo, C.; Zaccarelli, E.; Chiessi, E.; Corezzi, S. Thermoresponsivity of poly(N-isopropylacrylamide) microgels in water-trehalose solution and its relation to protein behavior. J. Colloid Interface Sci. 2021, 604, 705– 718,  DOI: 10.1016/j.jcis.2021.07.006
      12
      Thermoresponsivity of poly(N-isopropylacrylamide) microgels in water-trehalose solution and its relation to protein behavior
      Rosi, Benedetta Petra; Tavagnacco, Letizia; Comez, Lucia; Sassi, Paola; Ricci, Maria; Buratti, Elena; Bertoldo, Monica; Petrillo, Caterina; Zaccarelli, Emanuela; Chiessi, Ester; Corezzi, Silvia
      Journal of Colloid and Interface Science (2021), 604 (), 705-718CODEN: JCISA5; ISSN:0021-9797. (Elsevier B.V.)
      Additives are commonly used to tune macromol. conformational transitions. Among additives, trehalose is an excellent bioprotectant and among responsive polymers, PNIPAM is the most studied material. Nevertheless, their interaction mechanism so far has only been hinted without direct investigation, and, crucially, never elucidated in comparison to proteins. Detailed insights would help understand to what extent PNIPAM microgels can effectively be used as synthetic biomimetic materials, to reproduce and study, at the colloidal scale, isolated protein behavior and its sensitivity to interactions with specific cosolvents or cosolutes. The effect of trehalose on the swelling behavior of PNIPAM microgels was monitored by dynamic light scattering; Raman spectroscopy and mol. dynamics simulations were used to explore changes of solvation and dynamics across the swelling-deswelling transition at the mol. scale. Strongly hydrated trehalose mols. develop water-mediated interactions with PNIPAM microgels, thereby preserving polymer hydration below and above the transition while drastically inhibiting local motions of the polymer and of its hydration shell. Our study, for the first time, demonstrates that slowdown of dynamics and preferential exclusion are the principal mechanisms governing trehalose effect on PNIPAM microgels, at odds with preferential adsorption of alcs., but in full analogy with the behavior obsd. in trehalose-protein systems.
    13. 13
      Yan, X.; Chu, Y.; Liu, B.; Ru, G.; Di, Y.; Feng, J. Dynamic Mechanism of Halide Salts on the Phase Transition of Protein Models Poly(N-isopropylacrylamide) and Poly(N,N-diethylacrylamide). Phys. Chem. Chem. Phys. 2020, 22, 12644– 12650,  DOI: 10.1039/D0CP01366H
      13
      Dynamic mechanism of halide salts on the phase transition of protein models, poly(N-isopropylacrylamide) and poly(N,N-diethylacrylamide)
      Yan, Xiaoshuang; Chu, Yueying; Liu, Biaolan; Ru, Geying; Di, Yi; Feng, Jiwen
      Physical Chemistry Chemical Physics (2020), 22 (22), 12644-12650CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)
      The effects of salts on protein systems are not yet fully understood. We investigated the ionic dynamics of three halide salts (NaI, NaBr, and NaCl) with two protein models, namely poly(N-isopropylacrylamide) (PNIPAM) and poly(N,N-diethylacrylamide) (PDEA), using multinuclear NMR, dispersion cor. d. functional theory (DFT-D) calcns. and dynamic light scattering (DLS) methods. The variation in ionic line-widths and chem. shifts induced by the polymers clearly illustrates that anions rather than cations interact directly with the polymers. From the variable temp. measurements of the NMR transverse relaxation rates of anions, which characterize the polymer-anion interaction intensities, the evolution behaviors of Cl-/Br-/I- during phase transitions are similar in each polymer system but differ between the two polymer systems. The NMR transverse relaxation rates of anions change synchronously with the phase transition of PNIPAM upon heating, but they drop rapidly and vanish about 3-4.5° before the phase transition of PDEA. By combining the DFT-D and DLS data, the relaxation results imply that anions escape from the interacting sites with PDEA prior to full polymer dehydration or collapse, which can be attributed to the lack of anion-NH interactions. The different dynamic evolutions of the anions in the PNIPAM and PDEA systems give us an important clue for understanding the micro-mechanism of protein folding in a complex salt aq. solvent.
    14. 14
      Zhang, Y.; Furyk, S.; Bergbreiter, D. E.; Cremer, P. S. Specific Ion Effects on the Water Solubility of Macromolecules: PNIPAM and the Hofmeister Series. J. Am. Chem. Soc. 2005, 127, 14505– 14510,  DOI: 10.1021/ja0546424
      14
      Specific Ion Effects on the Water Solubility of Macromolecules: PNIPAM and the Hofmeister Series
      Zhang, Yanjie; Furyk, Steven; Bergbreiter, David E.; Cremer, Paul S.
      Journal of the American Chemical Society (2005), 127 (41), 14505-14510CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Aq. processes ranging from protein folding and enzyme turnover to colloidal ordering and macromol. pptn. are sensitive to the nature and concn. of the ions present in soln. Herein, the effect of a series of sodium salts on the lower crit. soln. temp. (LCST) of poly(N-isopropylacrylamide), PNIPAM, was investigated with a temp. gradient microfluidic device under a dark-field microscope. While the ability of a particular anion to lower the LCST generally followed the Hofmeister series, anal. of solvent isotope effects and of the changes in LCST with ion concn. and identity showed multiple mechanisms were at work. In solns. contg. sufficient concns. of strongly hydrated anions, the phase transition of PNIPAM was directly correlated with the hydration entropy of the anion. On the other hand, weakly hydrated anions were salted-out through surface tension effects and displayed improved hydration by direct ion binding.
    15. 15
      Wu, J.; Wang, X.; Wang, Q.; Lou, Z.; Li, S.; Zhu, Y.; Qin, L.; Wei, H. Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes (II). Chem. Soc. Rev. 2019, 48, 1004– 1076,  DOI: 10.1039/C8CS00457A
      15
      Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes (II)
      Wu, Jiangjiexing; Wang, Xiaoyu; Wang, Quan; Lou, Zhangping; Li, Sirong; Zhu, Yunyao; Qin, Li; Wei, Hui
      Chemical Society Reviews (2019), 48 (4), 1004-1076CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      Nanozymes are nanomaterials with enzyme-like characteristics (Chem. Soc. Rev., 2013, 42, 6060-6093). They have been developed to address the limitations of natural enzymes and conventional artificial enzymes. Along with the significant advances in nanotechnol., biotechnol., catalysis science, and computational design, great progress has been achieved in the field of nanozymes since the publication of the above-mentioned comprehensive review in 2013. To highlight these achievements, this review first discusses the types of nanozymes and their representative nanomaterials, together with the corresponding catalytic mechanisms whenever available. Then, it summarizes various strategies for modulating the activity and selectivity of nanozymes. After that, the broad applications from biomedical anal. and imaging to theranostics and environmental protection are covered. Finally, the current challenges faced by nanozymes are outlined and the future directions for advancing nanozyme research are suggested. The current review can help researchers know well the current status of nanozymes and may catalyze breakthroughs in this field.
    16. 16
      Xie, J.; Zhang, X.; Wang, H.; Zheng, H.; Huang, Y.; Xie, J. Analytical and environmental applications of nanoparticles as enzyme mimetics. Trends in Analytical Chemistry 2012, 39, 114– 129,  DOI: 10.1016/j.trac.2012.03.021
      16
      Analytical and environmental applications of nanoparticles as enzyme mimetics
      Xie, Jianxin; Zhang, Xiaodan; Wang, Hui; Zheng, Huzhi; Huang, Yuming; Xie, Jianxin
      TrAC, Trends in Analytical Chemistry (2012), 39 (), 114-129CODEN: TTAEDJ; ISSN:0165-9936. (Elsevier B. V.)
      A review. Recently, the intrinsic enzyme-like activity of nanoparticles (NPs) has become a growing area of interest. Compared with natural enzymes, these enzyme-like NPs are stable against denaturing, low in cost, and highly resistant to high concns. of substrate. These advantages make them promising in various applications. In this review, we focus on recent advances in NPs as enzyme mimetics and their anal. and environmental applications. We pay special attention to the enzyme-like activity of magnetic NPs, cerium-oxide NPs, noble-metal NPs, carbon and other nanomaterials.
    17. 17
      Vial, L.; Dumy, P. Artificial enzyme-based biosensors. New J. Chem. 2009, 33, 939– 946,  DOI: 10.1039/b822966j
      17
      Artificial enzyme-based biosensors
      Vial, Laurent; Dumy, Pascal
      New Journal of Chemistry (2009), 33 (5), 939-946CODEN: NJCHE5; ISSN:1144-0546. (Royal Society of Chemistry)
      A review. During the last forty years, enzyme-based biosensors have had great success in the detection and quantification of various biol.-relevant mols. However, native enzymes can sometimes be costly, delicate to manipulate or simply absent for a particular analyte. Hence, artificial or synthetic enzymes could be a useful alternative to natural proteins for the conception of new biosensors, since they can be, a priori, designed in their entirety, as well as more robust, available, chem. malleable and cheap, in comparison with their natural analogs. In this Perspective, the authors will provide a snapshot of this emerging research field.
    18. 18
      Halperin, A.; Kröger, M.; Winnik, F. M. Poly(N-isopropylacrylamide) Phase Diagrams: Fifty Years of Research. Angew. Chem., Int. Ed. 2015, 54, 15342– 15367,  DOI: 10.1002/anie.201506663
      18
      Poly(N-isopropylacrylamide) Phase Diagrams: Fifty Years of Research
      Halperin, Avraham; Kroeger, Martin; Winnik, Francoise M.
      Angewandte Chemie, International Edition (2015), 54 (51), 15342-15367CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. In 1968, Heskins and Guillet published the first systematic study of the phase diagram of poly(N-isopropylacrylamide) (PNIPAM), at the time a "young polymer" first synthesized in 1956. Since then, PNIPAM became the leading member of the growing families of thermoresponsive polymers and of stimuli-responsive, "smart" polymers in general. Its thermal response is unanimously attributed to its phase behavior. Yet, in spite of 50 years of research, a coherent quant. picture remains elusive. In this Review we survey the reported phase diagrams, discuss the differences and comment on theor. ideas regarding their possible origins. We aim to alert the PNIPAM community to open questions in this reputably mature domain.
    19. 19
      Tavagnacco, L.; Zanatta, M.; Buratti, E.; Rosi, B.; Frick, B.; Natali, F.; Ollivier, J.; Chiessi, E.; Bertoldo, M.; Zaccarelli, E.; Orecchini, A. Proteinlike dynamical transition of hydrated polymer chains. Physical Review Research 2021, 3, 013191,  DOI: 10.1103/PhysRevResearch.3.013191
      19
      Proteinlike dynamical transition of hydrated polymer chains
      Tavagnacco, L.; Zanatta, M.; Buratti, E.; Rosi, B.; Frick, B.; Natali, F.; Ollivier, J.; Chiessi, E.; Bertoldo, M.; Zaccarelli, E.; Orecchini, A.
      Physical Review Research (2021), 3 (1), 013191CODEN: PRRHAI; ISSN:2643-1564. (American Physical Society)
      Combining elastic incoherent neutron scattering expts. at different resolns. with mol. dynamics simulations, we report the observation of a proteinlike dynamical transition in linear chains of poly(N-isopropylacrylamide). We identify the onset of the transition at a temp. Td of about 225 K. Due to a global fit procedure, we find quant. agreement between measured and calcd. polymer mean-squared displacements at all temps. and time resolns. Our results confirm the generality of the dynamical transition in macromol. systems in aq. environments, independently of the internal polymer topol.
    20. 20
      Tavagnacco, L.; Chiessi, E.; Zanatta, M.; Orecchini, A.; Zaccarelli, E. Water–Polymer Coupling Induces a Dynamical Transition in Microgels. J. Phys. Chem. Lett. 2019, 10, 870– 876,  DOI: 10.1021/acs.jpclett.9b00190
      20
      Water-Polymer Coupling Induces a Dynamical Transition in Microgels
      Tavagnacco, Letizia; Chiessi, Ester; Zanatta, Marco; Orecchini, Andrea; Zaccarelli, Emanuela
      Journal of Physical Chemistry Letters (2019), 10 (4), 870-876CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
      The long debated protein dynamical transition was recently found also in nonbiol. macromols., such as poly-N-isopropylacrylamide (PNIPAM) microgels. Here, by using atomistic mol. dynamics simulations, we report a description of the mol. origin of the dynamical transition in these systems. We show that PNIPAM and water dynamics below the dynamical transition temp. Td are dominated by Me group rotations and hydrogen bonding, resp. By comparing with bulk water, we unambiguously identify PNIPAM-water hydrogen bonding as mainly responsible for the occurrence of the transition. The obsd. phenomenol. thus crucially depends on the water-macromol. coupling, being relevant to a wide class of hydrated systems, independently from the biol. function.
    21. 21
      Zanatta, M.; Tavagnacco, L.; Buratti, E.; Bertoldo, M.; Natali, F.; Chiessi, E.; Orecchini, A.; Zaccarelli, E. Evidence of a low-temperature dynamical transition in concentrated microgels. Science Advances 2018, 4, eaat5895  DOI: 10.1126/sciadv.aat5895
      There is no corresponding record for this reference.
    22. 22
      Doster, W.; Cusack, S.; Petry, W. Dynamical Transition of Myoglobin Revealed by Inelastic Neutron Scattering. Nature 1989, 337, 754– 6,  DOI: 10.1038/337754a0
      22
      Dynamical transition of myoglobin revealed by inelastic neutron scattering
      Doster, Wolfgang; Cusack, Stephen; Petry, Winfried
      Nature (London, United Kingdom) (1989), 337 (6209), 754-6CODEN: NATUAS; ISSN:0028-0836.
      The technique of inelastic neutron scattering was used to investigate at. motion in hydrated myoglobin over the temp. range 4-350 K and on the mol. dynamics timescale 0.1-100 ps. At temps. <180 K myoglobin behaves as a harmonic solid, with essentially only vibrational motion. Above 180 K there is a striking dynamic transition arising from the excitation of nonvibrational motion, which is interpreted as corresponding to torsional jumps between states of different energy, with a mean energy asymmetry of 12 kJ mol-1. This extra mobility is reflected in a strong temp. dependence of the mean-square at. displacements, a phenomenon previously obsd. specifically for the heme Fe by Moessbauer spectroscopy, but on a much slower timescale (10-7 s). It also correlates with a glass-like transition in the hydration shell of myoglobin and with the temp. dependence of ligand-binding rates at the heme Fe, as monitored by flash photolysis. The dynamical behavior found for myoglobin (and other globular proteins) suggests a coupling of fast local motions to slower collective motions, which is a characteristic feature of other dense glass-forming systems.
    23. 23
      Schiró, G.; Weik, M. Role of hydration water in the onset of protein structural dynamics. J. Phys.: Condens. Matter 2019, 31, 463002,  DOI: 10.1088/1361-648X/ab388a
      23
      Role of hydration water in the onset of protein structural dynamics
      Schiro, Giorgio; Weik, Martin
      Journal of Physics: Condensed Matter (2019), 31 (46), 463002CODEN: JCOMEL; ISSN:0953-8984. (IOP Publishing Ltd.)
      A review. Proteins are the mol. workhorses in a living organism. Their 3D structures are animated by a multitude of equil. fluctuations and specific out-of-equil. motions that are required for proteins to be biol. active. When studied as a function of temp., functionally relevant dynamics are obsd. at and above the so-called protein dynamical transition (∼240 K) in hydrated, but not in dry proteins. In this review we present and discuss the main exptl. and computational results that provided evidence for the dynamical transition, with a focus on the role of hydration water dynamics in sustaining functional protein dynamics. The coupling and mutual influence of hydration water dynamics and protein dynamics are discussed and the hypotheses illustrated that have been put forward to explain the phys. origin of their onsets.
    24. 24
      Cornicchi, E.; Capponi, S.; Marconi, M.; Onori, G.; Paciaroni, A. Temperature dependence of fast fluctuations in single- and double-stranded DNA molecules: a neutron scattering investigation. Philos. Mag. 2007, 87, 509– 515,  DOI: 10.1080/14786430600909022
      24
      Temperature dependence of fast fluctuations in single- and double-stranded DNA molecules: a neutron scattering investigation
      Cornicchi, E.; Capponi, S.; Marconi, M.; Onori, G.; Paciaroni, A.
      Philosophical Magazine (2007), 87 (3-5), 509-515CODEN: PMHABF; ISSN:1478-6435. (Taylor & Francis Ltd.)
      Using elastic neutron scattering measurements we have investigated the picosecond dynamics of dry and hydrated powders of DNA in the double-stranded (dsDNA) and single-stranded (ssDNA) state in the temp. range from 20 to 300 K. The extd. mean square displacements of DNA hydrogen atoms exhibit an onset of anharmonicity at around 100 K. The dynamics of the hydrated samples shows a further anharmonic contribution appearing at a temp. Td = 230-240 K. Such dynamical behavior is similar to the well-studied dynamical transition found in hydrated protein powders. The mean square displacements of dsDNA and ssDNA are practically superimposed in the whole temp. range for both dry and hydrated samples. This suggests that the DNA local mobility in the picosecond timescale does not depend on the single- or double-stranded conformation.
    25. 25
      Cornicchi, E.; De Francesco, A.; Marconi, M.; Onori, G.; Paciaroni, A. A relationship between solvent viscosity and biomolecule picosecond thermal fluctuations. Chem. Phys. 2008, 345, 219– 223,  DOI: 10.1016/j.chemphys.2007.07.022
      25
      A relationship between solvent viscosity and biomolecule picosecond thermal fluctuations
      Cornicchi, E.; De Francesco, A.; Marconi, M.; Onori, G.; Paciaroni, A.
      Chemical Physics (2008), 345 (2-3), 219-223CODEN: CMPHC2; ISSN:0301-0104. (Elsevier B.V.)
      Through elastic neutron scattering measurements, we investigated the picosecond dynamics of DNA in the hydrated powder state or embedded in glycerol glassy matrix from 20 K to 300 K. We calcd. the relaxational contribution of the mean square displacements (MSD) of DNA hydrogen atoms. We found the existence of a linear relationship between the inverse of the biomol. relaxational MSD and the logarithm of the bulk viscosity of the surrounding environment. From the comparison with the case of lysozyme in the same environments, for which the validity of the relationship was already verified, possible differences and analogies concerning the biomol.-to-solvent dynamical coupling can be stressed.
    26. 26
      Caliskan, G.; Briber, R. M.; Thirumalai, D.; García-Sakai, V.; Woodson, S. A.; Sokolov, A. P. Dynamic Transition in tRNA is Solvent Induced. J. Am. Chem. Soc. 2006, 128, 32– 33,  DOI: 10.1021/ja056444i
      26
      Dynamic Transition in tRNA is Solvent Induced
      Caliskan, Gokhan; Briber, Robert M.; Thirumalai, D.; Garcia-Sakai, Victoria; Woodson, Sarah A.; Sokolov, Alexei P.
      Journal of the American Chemical Society (2006), 128 (1), 32-33CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Dynamics of tRNA was studied using neutron scattering spectroscopy. Despite vast differences in the architecture and backbone structure of proteins and RNA, hydrated tRNA undergoes the dynamic transition at the same temp. as hydrated lysozyme. The similarity of the dynamic transition in RNA and proteins supports the idea that it is solvent induced. Because tRNA essentially has no Me groups, the results also suggest that Me groups are not the main contributor of the dynamic transition in biol. macromols. However, they may explain strong differences in the dynamics of tRNA and lysozyme obsd. at low temps.
    27. 27
      Roh, J.; Briber, R.; Damjanovic, A.; Thirumalai, D.; Woodson, S.; Sokolov, A. Dynamics of tRNA at Different Levels of Hydration. Biophys. J. 2009, 96, 2755– 2762,  DOI: 10.1016/j.bpj.2008.12.3895
      27
      Dynamics of tRNA at different levels of hydration
      Roh, J. H.; Briber, R. M.; Damjanovic, A.; Thirumalai, D.; Woodson, S. A.; Sokolov, A. P.
      Biophysical Journal (2009), 96 (7), 2755-2762CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      The influence of hydration on the nanosecond timescale dynamics of tRNA is investigated using neutron scattering spectroscopy. Unlike protein dynamics, the dynamics of tRNA is not affected by Me group rotation. This allows for a simpler anal. of the influence of hydration on the conformational motions in RNA. We find that hydration affects the dynamics of tRNA significantly more than that of lysozyme. Both the characteristic length scale and the timescale of the conformational motions in tRNA depend strongly on hydration. Even the characteristic temp. of the so-called "dynamical transition" appears to be hydration-dependent in tRNA. The amplitude of the conformational motions in fully hydrated tRNA is almost twice as large as in hydrated lysozyme. We ascribe these differences to a more open and flexible structure of hydrated RNA, and to a larger fraction and different nature of hydrophilic sites. The latter leads to a higher d. of water that makes the biomol. more flexible. All-atom mol.-dynamics simulations are used to show that the extent of hydration is greater in tRNA than in lysozyme. We propose that water acts as a "lubricant" in facilitating enhanced motion in solvated RNA mols.
    28. 28
      Capaccioli, S.; Ngai, K. L.; Ancherbak, S.; Paciaroni, A. Evidence of Coexistence of Change of Caged Dynamics at Tg and the Dynamic Transition at Td in Solvated Proteins. J. Phys. Chem. B 2012, 116, 1745– 1757,  DOI: 10.1021/jp2057892
      28
      Evidence of Coexistence of Change of Caged Dynamics at Tg and the Dynamic Transition at Td in Solvated Proteins
      Capaccioli, S.; Ngai, K. L.; Ancherbak, S.; Paciaroni, A.
      Journal of Physical Chemistry B (2012), 116 (6), 1745-1757CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
      Moessbauer spectroscopy and neutron scattering measurements on proteins embedded in solvents including water and aq. mixts. have emphasized the observation of the distinctive temp. dependence of the at. mean square displacements, 〈U2〉, commonly referred to as the dynamic transition at some temp. Td. At low temps., 〈U2〉 increases slowly, but it assumes stronger temp. dependence after crossing Td, which depends on the time/frequency resoln. of the spectrometer. Various authors have made connection of the dynamics of solvated proteins, including the dynamic transition to that of glass-forming substances. Notwithstanding, no connection is made to the similar change of temp. dependence of 〈U2〉 obtained by quasielastic neutron scattering when crossing the glass transition temp. Tg, generally obsd. in inorg., org., and polymeric glass-formers. Evidences are presented here to show that such a change of the temp. dependence of 〈U2〉 from neutron scattering at Tg is present in hydrated or solvated proteins, as well as in the solvent used, unsurprisingly since the latter is just another org. glass-former. If unaware of the existence of such a crossover of 〈U2〉 at Tg, and if present, it can be mistaken as the dynamic transition at Td with the ill consequences of underestimating Td by the lower value Tg and confusing the identification of the origin of the dynamic transition. The 〈U2〉 obtained by neutron scattering at not so low temps. has contributions from the dissipation of mols. while caged by the anharmonic intermol. potential at times before dissoln. of cages by the onset of the Johari-Goldstein β-relaxation or of the merged α-β relaxation. The universal change of 〈U2〉 at Tg of glass-formers, independent of the spectrometer resoln., had been rationalized by sensitivity to change in vol. and entropy of the dissipation of the caged mols. and its contribution to 〈U2〉. The same rationalization applies to hydrated and solvated proteins for the obsd. change of 〈U2〉 at Tg.
    29. 29
      Ngai, K.; Capaccioli, S.; Paciaroni, A. Dynamics of hydrated proteins and bio-protectants: Caged dynamics, β-relaxation, and α-relaxation. Biochimica et Biophysica Acta (BBA) - General Subjects 2017, 1861, 3553– 3563,  DOI: 10.1016/j.bbagen.2016.04.027
      There is no corresponding record for this reference.
    30. 30
      Chen, S.-H.; Liu, L.; Fratini, E.; Baglioni, P.; Faraone, A.; Mamontov, E. Observation of fragile-to-strong dynamic crossover in protein hydration water. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 9012– 9016,  DOI: 10.1073/pnas.0602474103
      30
      Observation of fragile-to-strong dynamic crossover in protein hydration water
      Chen, S.-H.; Liu, L.; Fratini, E.; Baglioni, P.; Faraone, A.; Mamontov, E.
      Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (24), 9012-9016CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      At low temps., proteins exist in a glassy state, a state that has no conformational flexibility and shows no biol. functions. In a hydrated protein, at temps. ≥220 K, this flexibility is restored, and the protein is able to sample more conformational substates, thus becoming biol. functional. This "dynamical" transition of protein is believed to be triggered by its strong coupling with the hydration water, which also shows a similar dynamic transition. Here we demonstrate exptl. that this sudden switch in dynamic behavior of the hydration water on lysozyme occurs precisely at 220 K and can be described as a fragile-to-strong dynamic crossover. At the fragile-to-strong dynamic crossover (FSC), the structure of hydration water makes a transition from predominantly high-d. (more fluid state) to low-d. (less fluid state) forms derived from the existence of the second crit. point at an elevated pressure.
    31. 31
      Wood, K.; Frölich, A.; Paciaroni, A.; Moulin, M.; Härtlein, M.; Zaccai, G.; Tobias, D. J.; Weik, M. Coincidence of Dynamical Transitions in a Soluble Protein and Its Hydration Water: Direct Measurements by Neutron Scattering and MD Simulations. J. Am. Chem. Soc. 2008, 130, 4586– 4587,  DOI: 10.1021/ja710526r
      31
      Coincidence of Dynamical Transitions in a Soluble Protein and Its Hydration Water: Direct Measurements by Neutron Scattering and MD Simulations
      Wood, Kathleen; Froelich, Andreas; Paciaroni, Alessandro; Moulin, Martine; Haertlein, Michael; Zaccai, Giuseppe; Tobias, Douglas J.; Weik, Martin
      Journal of the American Chemical Society (2008), 130 (14), 4586-4587CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The coupling between protein dynamics and hydration-water dynamics was assessed by perdeuteration, temp.-dependent neutron scattering, and mol. dynamics simulations. Mean square displacements of water and protein motions both show a broad transition at 220 K and are thus coupled. In particular, the protein dynamical transition appears to be driven by the onset of hydration-water translational motion.
    32. 32
      Schiró, G.; Vetri, V.; Frick, B.; Militello, V.; Leone, M.; Cupane, A. Neutron Scattering Reveals Enhanced Protein Dynamics in Concanavalin A Amyloid Fibrils. J. Phys. Chem. Lett. 2012, 3, 992– 996,  DOI: 10.1021/jz300082x
      32
      Neutron scattering reveals enhanced protein dynamics in concanavalin A amyloid fibrils
      Schiro, Giorgio; Vetri, Valeria; Frick, Bernhard; Militello, Valeria; Leone, Maurizio; Cupane, Antonio
      Journal of Physical Chemistry Letters (2012), 3 (8), 992-996CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
      Protein aggregation is one of the most challenging topics in life sciences, and it is implicated in several human pathologies. The nature and the role of toxic species is highly debated, with amyloid fibrils being among the most relevant species for their peculiar structural and functional properties. Protein dynamics and in particular the ability to fluctuate through a large no. of conformational substates are closely related to protein function. Here, the authors focus on amyloid fibril dynamics, and, to their knowledge, it is the 1st neutron scattering study on a protein (Con A) isolated in its fibril state. The results revealed enhanced at. fluctuations in amyloid fibrils and indicated that the protein was "softer" in the fibril state with respect to the native and amorphous aggregate states. The authors discuss this finding in terms of a structural interpretation and suggest that the paradigm ordered structure ↔ lower flexibility can be questioned when considering the local fast side-chain protein dynamics.
    33. 33
      Schiró, G.; Caronna, C.; Natali, F.; Koza, M. M.; Cupane, A. The “Protein Dynamical Transition” Does Not Require the Protein Polypeptide Chain. J. Phys. Chem. Lett. 2011, 2, 2275– 2279,  DOI: 10.1021/jz200797g
      33
      The "protein dynamical transition" does not require the protein polypeptide chain
      Schiro, Giorgio; Caronna, Chiara; Natali, Francesca; Koza, M. Marek; Cupane, Antonio
      Journal of Physical Chemistry Letters (2011), 2 (18), 2275-2279CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
      The authors provide exptl. evidence that the main features of protein dynamics revealed by neutron scattering, i.e., the "protein dynamical transition" and the "boson peak", do not require the protein polypeptide chain. The authors show that a rapid increase of H atom fluctuations at ∼220 K, analogous to the one obsd. in hydrated myoglobin powders, was also obsd. in a mixt. of hydrated amino acids with the chem. compn. of myoglobin but lacking the polypeptide chain; in agreement with the protein behavior, the transition was abolished in the dry mixt. Further, an excess of low-frequency vibrational modes at ∼3 meV, typically obsd. in protein powders, was also obsd. in the authors' mixt. These results confirmed that the dynamical transition is a water-driven onset and indicated that it mainly involves the amino acid side chains. Taking together the present data and recent results on the dynamics of a protein in denatured conformation and on the activity of dehydrated proteins, it could be concluded that the "protein dynamical transition" is neither a necessary nor a sufficient condition for active protein conformation and function.
    34. 34
      Niessen, K. A.; Xu, M.; Paciaroni, A.; Orecchini, A.; Snell, E. H.; Markelz, A. G. Moving in the Right Direction: Protein Vibrations Steering Function. Biophys. J. 2017, 112, 933– 942,  DOI: 10.1016/j.bpj.2016.12.049
      34
      Moving in the Right Direction: Protein Vibrations Steering Function
      Niessen, Katherine A.; Xu, Mengyang; Paciaroni, Alessandro; Orecchini, Andrea; Snell, Edward H.; Markelz, Andrea G.
      Biophysical Journal (2017), 112 (5), 933-942CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      Nearly all protein functions require structural change, such as enzymes clamping onto substrates, and ion channels opening and closing. These motions are a target for possible new therapies; however, the control mechanisms are under debate. Calcns. have indicated protein vibrations enable structural change. However, previous measurements found these vibrations only weakly depend on the functional state. By using the novel technique of anisotropic terahertz microscopy (ATM), we find that there is a dramatic change to the vibrational directionality with inhibitor (3NAG) binding to lysozyme, whereas the vibrational energy distribution, as measured by neutron inelastic scattering, is only slightly altered. The anisotropic terahertz measurements provide unique access to the directionality of the intramol. vibrations, and immediately resolve the inconsistency between calcns. and previous measurements, which were only sensitive to the energy distribution. The biol. importance of the vibrational directions vs. the energy distribution is revealed by our calcns. comparing wild-type lysozyme with a higher catalytic rate double deletion mutant. The vibrational energy distribution is identical, but the more efficient mutant shows an obvious reorientation of motions. These results show that it is essential to characterize the directionality of motion to understand and control protein dynamics to optimize or inhibit function.
    35. 35
      Henzler-Wildman, K.; Kern, D. Dynamic personalities of proteins. Nature 2007, 450, 964– 972,  DOI: 10.1038/nature06522
      35
      Dynamic personalities of proteins
      Henzler-Wildman, Katherine; Kern, Dorothee
      Nature (London, United Kingdom) (2007), 450 (7172), 964-972CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      A review. Because proteins are central to cellular function, researchers have sought to uncover the secrets of how these complex macromols. execute such a fascinating variety of functions. Although static structures are known for many proteins, the functions of proteins are governed ultimately by their dynamic character (or 'personality'). The dream is to 'watch' proteins in action in real time at at. resoln. This requires the addn. of a fourth dimension, time, to structural biol. so that the positions in space and time of all atoms in a protein can be described in detail.
    36. 36
      Fenimore, P. W.; Frauenfelder, H.; McMahon, B. H.; Young, R. D. Bulk-solvent and hydration-shell fluctuations, similar to α- and β-fluctuations in glasses, control protein motions and functions. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 14408– 14413,  DOI: 10.1073/pnas.0405573101
      36
      Bulk-solvent and hydration-shell fluctuations, similar to α- and β-fluctuations in glasses, control protein motions and functions
      Fenimore, P. W.; Frauenfelder, Hans; McMahon, B. H.; Young, R. D.
      Proceedings of the National Academy of Sciences of the United States of America (2004), 101 (40), 14408-14413CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The concept that proteins exist in numerous different conformations or conformational substates, described by an energy landscape, is now accepted, but the dynamics is incompletely explored. We have previously shown that large-scale protein motions, such as the exit of a ligand from the protein interior, follow the dielec. fluctuations in the bulk solvent. Here, we demonstrate, by using mean-square displacements (msd) from Moessbauer and neutron-scattering expts., that fluctuations in the hydration shell control fast fluctuations in the protein. We call the first type solvent-slaved or α-fluctuations and the second type hydration-shell-coupled or β-fluctuations. Solvent-slaved motions are similar to the α-fluctuations in glasses. Their temp. dependence can be approximated by a Vogel-Tammann-Fulcher relation and they are absent in a solid environment. Hydration-shell-coupled fluctuations are similar to the β-relaxation in glasses. They can be approximated by a Ferry or an Arrhenius relation, are much reduced or absent in dehydrated proteins, and occur in hydrated proteins even if embedded in a solid. They can be responsible for internal processes such as the migration of ligands within myoglobin. The existence of two functionally important fluctuations in proteins, one slaved to bulk motions and the other coupled to hydration-shell fluctuations, implies that the environment can control protein functions through different avenues and that no real protein transition occurs at ≈200 K. The large no. of conformational substates is essential; proteins cannot function without this reservoir of entropy, which resides mainly in the hydration shell.
    37. 37
      Rasmussen, B. F.; Stock, A. M.; Ringe, D.; Petsko, G. A. Crystalline Ribonuclease A Loses Function Below the Dynamical Transition at 220 K. Nature 1992, 357, 423– 424,  DOI: 10.1038/357423a0
      37
      Crystalline ribonuclease A loses function below the dynamical transition at 220 K
      Rasmussen, Bjarne F.; Stock, Ann M.; Ringe, Dagmar; Petsko, Gregory A.
      Nature (London, United Kingdom) (1992), 357 (6377), 423-4CODEN: NATUAS; ISSN:0028-0836.
      When the dynamic properties of many different proteins are plotted as a function of temp., biphasic behavior is obsd., with a broad transition centered around 220 K. Mol. dynamics simulations over a range of temps. also exhibit a transition at about 220 K. Here, high-resoln. x-ray diffraction was used to show that cryst. RNase A does not bind substrate or inhibitor at 212 K but will bind either rapidly at 228 K. Once bound at the higher temp., inhibitor cannot be washed off after the enzyme is cooled to below the transition temp. These results suggest that enzyme flexibility is required for catalytic function.
    38. 38
      Réat, V.; Patzelt, H.; Ferrand, M.; Pfister, C.; Oesterhelt, D.; Zaccai, G. Dynamics of different functional parts of bacteriorhodopsin: H-2H labeling and neutron scattering. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 4970– 4975,  DOI: 10.1073/pnas.95.9.4970
      38
      Dynamics of different functional parts of bacteriorhodopsin: H-2H labeling and neutron scattering
      Reat, Valerie; Patzelt, Heiko; Ferrand, Michel; Pfister, Claude; Oesterhelt, Dieter; Zaccai, Giuseppe
      Proceedings of the National Academy of Sciences of the United States of America (1998), 95 (9), 4970-4975CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      We show that dynamics of specific amino acids within a protein can be characterized by neutron spectroscopy and hydrogen-deuterium labeling, and we present data on the motions of a selected set of groups within bacteriorhodopsin (BR), the retinal-based proton pump in the purple membrane of halophilic Archaea. Elastic incoherent neutron scattering expts. allow the definition of motions in the nano- to picosecond time scale and have revealed a dynamical transition from a harmonic to a softer, anharmonic at. fluctuation regime in the global behavior of proteins. Biol. activity in proteins is correlated with this transition, suggesting that flexibility is required for function. Elastic incoherent neutron scattering is dominated by H atom scattering, and to study the dynamics of a selected part of BR, fully deuterated purple membrane with BR contg. H-retinal, H-tryptophan, and H-methionine was prepd. biosynthetically in Halobacterium salinarum. These amino acids cluster in the functional center of the protein. In contrast to the protein globally, the thermal motions of the labeled atoms were found to be shielded from solvent melting effects at 260 K. Above this temp., the labeled groups appear as more rigid than the rest of the protein, with a significantly smaller mean square amplitude of motion. These exptl. results quantify the dynamical heterogeneity of BR (which meets the functional requirements of global flexibility), on the one hand, to allow large conformational changes in the mol. and of a more rigid region in the protein, on the other, to control stereo-specific selection of retinal conformations.
    39. 39
      Mensink, M. A.; Frijlink, H. W.; van der Voort Maarschalk, K.; Hinrichs, W. L. How sugars protect proteins in the solid state and during drying (review): Mechanisms of stabilization in relation to stress conditions. Eur. J. Pharm. Biopharm. 2017, 114, 288– 295,  DOI: 10.1016/j.ejpb.2017.01.024
      39
      How sugars protect proteins in the solid state and during drying (review): Mechanisms of stabilization in relation to stress conditions
      Mensink, Maarten A.; Frijlink, Henderik W.; van der Voort Maarschalk, Kees; Hinrichs, Wouter L. J.
      European Journal of Pharmaceutics and Biopharmaceutics (2017), 114 (), 288-295CODEN: EJPBEL; ISSN:0939-6411. (Elsevier B.V.)
      This review aims to provide an overview of current knowledge on stabilization of proteins by sugars in the solid state in relation to stress conditions commonly encountered during drying and storage. First protein degrdn. mechanisms in the solid state (i.e. phys. and chem. degrdn. routes) and traditional theories regarding protein stabilization (vitrification and water replacement hypotheses) will be briefly discussed. Secondly, refinements to these theories, such as theories focusing on local mobility and protein-sugar packing d., are reviewed in relationship to the traditional theories and their analogies are discussed. The last section relates these mechanistic insights to the stress conditions against which these sugars are used to provide protection (i.e. drying, temp., and moisture). In summary sugars should be able to adequately form interactions with the protein during drying, thereby maintaining it in its native conformation and reducing both local and global mobility during storage. Generally smaller sugars (disaccharides) are better at forming these interactions and reducing local mobility as they are less inhibited by steric hindrance, while larger sugars can reduce global mobility more efficiently. The principles outlined here can aid in choosing a suitable sugar as stabilizer depending on the protein, formulation and storage condition-specific dominant route of degrdn.
    40. 40
      Cicerone, M. T.; Pikal, M. J.; Qian, K. K. Stabilization of proteins in solid form. Adv. Drug Delivery Rev. 2015, 93, 14– 24,  DOI: 10.1016/j.addr.2015.05.006
      40
      Stabilization of proteins in solid form
      Cicerone, Marcus T.; Pikal, Michael J.; Qian, Ken K.
      Advanced Drug Delivery Reviews (2015), 93 (), 14-24CODEN: ADDREP; ISSN:0169-409X. (Elsevier B.V.)
      Immunogenicity of aggregated or otherwise degraded protein delivered from depots or other biopharmaceutical products is an increasing concern, and the ability to deliver stable, active protein is of central importance. We review characterization approaches for solid protein dosage forms with respect to metrics that are intended to be predictive of protein stability against aggregation and other degrdn. processes. Each of these approaches is ultimately motivated by hypothetical connections between protein stability and the material property being measured. We critically evaluate correlations between these properties and stability outcomes, and use these evaluations to revise the currently standing hypotheses. Based on this we provide simple phys. principles that are necessary (and possibly sufficient) for generating solid delivery vehicles with stable protein loads. Essentially, proteins should be strongly coupled (typically through H-bonds) to the bulk regions of a phase-homogeneous matrix with suppressed β relaxation. We also provide a framework for reliable characterization of solid protein forms with respect to stability.
    41. 41
      Manning, M.; Chou, D.; Murphy, B.; Payne, R.; Katayama, D. Stability of Protein Pharmaceuticals: An Update. Pharm. Res. 2010, 27, 544– 575,  DOI: 10.1007/s11095-009-0045-6
      41
      Stability of protein pharmaceuticals: an update
      Manning Mark Cornell; Chou Danny K; Murphy Brian M; Payne Robert W; Katayama Derrick S
      Pharmaceutical research (2010), 27 (4), 544-75 ISSN:.
      In 1989, Manning, Patel, and Borchardt wrote a review of protein stability (Manning et al., Pharm. Res. 6:903-918, 1989), which has been widely referenced ever since. At the time, recombinant protein therapy was still in its infancy. This review summarizes the advances that have been made since then regarding protein stabilization and formulation. In addition to a discussion of the current understanding of chemical and physical instability, sections are included on stabilization in aqueous solution and the dried state, the use of chemical modification and mutagenesis to improve stability, and the interrelationship between chemical and physical instability.
    42. 42
      Cordone, L.; Ferrand, M.; Vitrano, E.; Zaccai, G. Harmonic Behavior of Trehalose-Coated Carbon-Monoxy-Myoglobin at High Temperature. Biophys. J. 1999, 76, 1043– 1047,  DOI: 10.1016/S0006-3495(99)77269-3
      42
      Harmonic behavior of trehalose-coated carbon-monoxy-myoglobin at high temperature
      Cordone, Lorenzo; Ferrand, Michel; Vitrano, Eugenio; Zaccai, Giuseppe
      Biophysical Journal (1999), 76 (2), 1043-1047CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      Embedding biostructures in saccharide glasses protects them against extreme dehydration and/or exposure to very high temp. Among the saccharides, trehalose appears to be the most effective bioprotectant. In this paper we report on the low-frequency dynamics of carbon monoxy myoglobin in an extremely dry trehalose glass measured by neutron spectroscopy. Under these conditions, the mean square displacements and the d. of state function are those of a harmonic solid, up to room temp., in contrast to D2O-hydrated myoglobin, in which a dynamical transition to a nonharmonic regime has been obsd. at ∼180 K (Doster et al., 1989. Nature. 337:754-756). The protective effect of trehalose is correlated, therefore, with a trapping of the protein in a harmonic potential, even at relatively high temp.
    43. 43
      Cornicchi, E.; Marconi, M.; Onori, G.; Paciaroni, A. Controlling the Protein Dynamical Transition with Sugar-Based Bioprotectant Matrices: A Neutron Scattering Study. Biophys. J. 2006, 91, 289– 297,  DOI: 10.1529/biophysj.106.081752
      43
      Controlling the protein dynamical transition with sugar-based bioprotectant matrices: a neutron scattering study
      Cornicchi, E.; Marconi, M.; Onori, G.; Paciaroni, A.
      Biophysical Journal (2006), 91 (1), 289-297CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      Through elastic neutron scattering we measured the mean-square displacements (MSDs) of the hydrogen atoms of lysozyme embedded in a glucose-water glassy matrix as a function of the temp. and at various water contents. The elastic intensity of all the samples has been interpreted in terms of the double-well model in the whole temp. range. The dry sample shows an onset of anharmonicity at ∼100 K, which can be attributed to the activation of Me group reorientations. Such a protein intrinsic dynamics is decoupled from the external environment on the whole investigated temp. range. In the hydrated samples an addnl. and larger anharmonic contribution is provided by the protein dynamical transition, which appears at a higher temp. Td. As hydration increases the coupling between the protein internal dynamics and the surrounding matrix relaxations becomes more effective. The behavior of Td that, as a function of the water content, diminishes by ∼60 K, supports the picture of the protein dynamics as driven by solvent relaxations. A possible connection between the protein dynamical response vs. T and the thermal stability in glucose-water bioprotectant matrixes is proposed.
    44. 44
      Paciaroni, A.; Cinelli, S.; Onori, G. Effect of the Environment on the Protein Dynamical Transition: A Neutron Scattering Study. Biophys. J. 2002, 83, 1157– 1164,  DOI: 10.1016/S0006-3495(02)75239-9
      44
      Effect of the environment on the protein dynamical transition: a neutron scattering study
      Paciaroni, Alessandro; Cinelli, Stefania; Onori, Giuseppe
      Biophysical Journal (2002), 83 (2), 1157-1164CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      The authors performed an elastic neutron scattering investigation of the mol. dynamics of lysozyme solvated in glycerol, at different water contents h (grams of water/g of lysozyme). The marked nonGaussian behavior of the elastic intensity was studied in a wide exptl. momentum transfer range, as a function of the temp. The internal dynamics is well described in terms of the double-well jump model. At low temp., the protein total mean square displacements exhibit an almost linear harmonic trend irresp. of the hydration level, whereas at the temp. Td a clear changeover toward an anharmonic regime marks a protein dynamical transition. The decrease of Td from ∼238 K to ∼195 K as a function of h is reminiscent of that found in the glass transition temp. of aq. solns. of glycerol, thus suggesting that the protein internal dynamics as a whole is slave to the environment properties. Both Td and the total mean square displacements indicate that the protein flexibility strongly rises between 0.1 and 0.2h. This hydration-dependent dynamical activation, which is similar to that of hydrated lysozyme powders, is related to the specific interplay of the protein with the surrounding water and glycerol mols.
    45. 45
      Katava, M.; Stirnemann, G.; Zanatta, M.; Capaccioli, S.; Pachetti, M.; Ngai, K. L.; Sterpone, F.; Paciaroni, A. Critical structural fluctuations of proteins upon thermal unfolding challenge the Lindemann criterion. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 9361– 9366,  DOI: 10.1073/pnas.1707357114
      45
      Critical structural fluctuations of proteins upon thermal unfolding challenge the Lindemann criterion
      Katava, Marina; Stirnemann, Guillaume; Zanatta, Marco; Capaccioli, Simone; Pachetti, Maria; Ngai, K. L.; Sterpone, Fabio; Paciaroni, Alessandro
      Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (35), 9361-9366CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Internal subnanosecond timescale motions are key for the function of proteins, and are coupled to the surrounding solvent environment. These fast fluctuations guide protein conformational changes, yet their role for protein stability, and for unfolding, remains elusive. Here, in analogy with the Lindemann criterion for the melting of solids, we demonstrate a common scaling of structural fluctuations of lysozyme protein embedded in different environments as the thermal unfolding transition is approached. By combining elastic incoherent neutron scattering and advanced mol. simulations, we show that, although different solvents modify the protein melting temp., a unique dynamical regime is attained in proximity of thermal unfolding in all solvents that we tested. This solvation shell-independent dynamical regime arises from an equiv. sampling of the energy landscape at the resp. melting temps. Thus, we propose that a threshold for the conformational entropy provided by structural fluctuations of proteins exists, beyond which thermal unfolding is triggered.
    46. 46
      Marconi, M.; De Francesco, A.; Cornicchi, E.; Onori, G.; Paciaroni, A. Hydration and temperature dependent dynamics of lysozyme in glucose–water matrices. A neutron scattering study. Chem. Phys. 2005, 317, 274– 281,  DOI: 10.1016/j.chemphys.2005.06.027
      46
      Hydration and temperature dependent dynamics of lysozyme in glucose-water matrices. A neutron scattering study
      Marconi, M.; De Francesco, A.; Cornicchi, E.; Onori, G.; Paciaroni, A.
      Chemical Physics (2005), 317 (2-3), 274-281CODEN: CMPHC2; ISSN:0301-0104. (Elsevier B.V.)
      Through a neutron scattering expt., we studied the dynamics of lysozyme embedded in glucose-water matrixes. Two distinct quasielastic contributions, corresponding to motions in the sub- and picosecond timescales, are revealed after the subtraction of the inelastic intensity, as estd. at low temp. Their characteristic times are hydration and temp. independent, and quite similar to those revealed when the environment around the biomol. surface is pure water or glycerol. The momentum transfer dependence of each quasielastic signal has been interpreted in terms of the rotational diffusion model. The faster component corresponds to a more localized rotational motion on a radius of 1 Å, while the slower contribution is assocd. to a larger radius of 1.9 Å. The behavior of the fraction of moving protein hydrogen atoms as a function of temp. and hydration suggests that the protein undergoes a dynamical transition just at conditions corresponding to the dynamical onset of the enclosing matrix.
    47. 47
      Paciaroni, A.; Cornicchi, E.; De Francesco, A.; Marconi, M.; Onori, G. Conditioning action of the environment on the protein dynamics studied through elastic neutron scattering. Eur. Biophys. J. 2006, 35, 591– 599,  DOI: 10.1007/s00249-006-0073-7
      47
      Conditioning action of the environment on the protein dynamics studied through elastic neutron scattering
      Paciaroni, A.; Cornicchi, E.; De Francesco, A.; Marconi, M.; Onori, G.
      European Biophysics Journal (2006), 35 (7), 591-599CODEN: EBJOE8; ISSN:0175-7571. (Springer)
      The dynamics of lysozyme in the picosecond timescale was studied when it is in dry and hydrated powder form and when it is embedded in glycerol, glycerol-water, glucose and glucose-water matrixes. The investigation was undertaken through elastic neutron scattering technique on the backscattering spectrometer IN13. The dynamics of dry powder and embedded-in-glucose lysozyme can be considered purely vibrational up to 100 K, where the onset of an anharmonic contribution takes place. This contribution can be attributed to the activation of Me group reorientations and is described with an Arrhenius trend. An addnl. source of anharmonic dynamics appears at higher temps. for lysozyme in hydrated powders and embedded in glycerol, glycerol-water and glucose-water matrixes. This second process, also represented with an Arrhenius trend, corresponds to the so-called protein dynamical transition. Both the temp. where such a transition takes place and the magnitude of the protein mean square displacements depend on the environment. The dynamical response of the protein to temp. is put in relationship with its thermal stability.
    48. 48
      Tsai, A. M.; Neumann, D. A.; Bell, L. N. Molecular Dynamics of Solid-State Lysozyme as Affected by Glycerol and Water: A Neutron Scattering Study. Biophys. J. 2000, 79, 2728– 2732,  DOI: 10.1016/S0006-3495(00)76511-8
      48
      Molecular dynamics of solid-state lysozyme as affected by glycerol and water: a neutron scattering study
      Tsai, Amos M.; Neumann, Dan A.; Bell, Leonard N.
      Biophysical Journal (2000), 79 (5), 2728-2732CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      Glycerol has previously been shown to lower the heat denaturation temp. (Tm) of dehydrated lysozyme while elevating the Tm of hydrated lysozyme. Here, the authors report an in situ elastic neutron scattering study of the effect of glycerol and hydration on the internal dynamics of lysozyme powder. Anharmonic motions assocd. with structural relaxation processes were not detected for dehydrated lysozyme in the temp. range of 40-450K. Dehydrated lysozyme was previously found to have the highest Tm. Upon the addn. of glycerol or water, anharmonicity was recovered above a dynamic transition temp. (Td), which may contribute to the redn. of Tm values for dehydrated lysozyme in the presence of glycerol. The greatest degree of anharmonicity, as well as the lowest Td, was obsd. for lysozyme solvated with water. Hydrated lysozyme was also previously found to have the lowest Tm. In the regime above the Td, larger amts. of glycerol led to a higher rate of change in anharmonic motions as a function of temp., rendering the material more heat-labile. Below the Td, where harmonic motions dominate, the addn. of glycerol resulted in a lower amplitude of motions, correlating with a stabilizing effect of glycerol on the protein.
    49. 49
      Tsai, A. M.; Udovic, T. J.; Neumann, D. A. The Inverse Relationship between Protein Dynamics and Thermal Stability. Biophys. J. 2001, 81, 2339– 2343,  DOI: 10.1016/S0006-3495(01)75880-8
      49
      The inverse relationship between protein dynamics and thermal stability
      Tsai, Amos M.; Udovic, Terrence J.; Neumann, Dan A.
      Biophysical Journal (2001), 81 (4), 2339-2343CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      Protein powders that are dehydrated or mixed with a glassy compd. are known to have improved thermal stability. We present elastic and quasielastic neutron scattering measurements of the global dynamics of lysozyme and RNase A powders. In the absence of solvation water, both protein powders exhibit largely harmonic motions on the timescale of the measurements. Upon partial hydration, quasielastic scattering indicative of relaxational processes appears at sufficiently high temp. When the scattering spectrum are analyzed with the Kohlrausch-Williams-Watts formalism, the exponent β decreases with increasing temp., suggesting that multiple relaxation modes are emerging. When lysozyme was mixed with glycerol, its β values were higher than the hydrated sample at comparable temps., reflecting the viscosity and stabilizing effects of glycerol.
    50. 50
      Weng, L.; Stott, S. L.; Toner, M. Exploring Dynamics and Structure of Biomolecules, Cryoprotectants, and Water Using Molecular Dynamics Simulations: Implications for Biostabilization and Biopreservation. Annu. Rev. Biomed. Eng. 2019, 21, 1– 31,  DOI: 10.1146/annurev-bioeng-060418-052130
      50
      Exploring Dynamics and Structure of Biomolecules, Cryoprotectants, and Water Using Molecular Dynamics Simulations: Implications for Biostabilization and Biopreservation
      Weng, Lindong; Stott, Shannon L.; Toner, Mehmet
      Annual Review of Biomedical Engineering (2019), 21 (), 1-31CODEN: ARBEF7; ISSN:1523-9829. (Annual Reviews)
      Successful stabilization and preservation of biol. materials often utilize low temps. and dehydration to arrest mol. motion. Cryoprotectants are routinely employed to help the biol. entities survive the physicochem. and mech. stresses induced by cold or dryness. Mol. interactions between biomols., cryoprotectants, and water fundamentally det. the outcomes of preservation. The optimization of assays using the empirical approach is often limited in structural and temporal resoln., whereas classical mol. dynamics simulations can provide a cost-effective glimpse into the at.-level structure and interaction of individual mols. that dictate macroscopic behavior. Computational research on biomols., cryoprotectants, and water has provided invaluable insights into the development of new cryoprotectants and the optimization of preservation methods. We describe the rapidly evolving state of the art of mol. simulations of these complex systems, summarize the mol.-scale protective and stabilizing mechanisms, and discuss the challenges that motivate continued innovation in this field.
    51. 51
      Cicerone, M. T.; Soles, C. L. Fast Dynamics and Stabilization of Proteins: Binary Glasses of Trehalose and Glycerol. Biophys. J. 2004, 86, 3836– 3845,  DOI: 10.1529/biophysj.103.035519
      51
      Fast dynamics and stabilization of proteins: Binary glasses of trehalose and glycerol
      Cicerone, Marcus T.; Soles, Christopher L.
      Biophysical Journal (2004), 86 (6), 3836-3845CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      We present elastic and inelastic incoherent neutron scattering data from a series of trehalose glasses dild. with glycerol. A strong correlation with recently published protein stability data in the same series of glasses illustrates that the dynamics at Q ≥ 0.71 Å-1 and ω > 200 MHz are important to stabilization of horseradish peroxidase and yeast alc. dehydrogenase in these glasses. To the best of our knowledge, this is the first direct evidence that enzyme stability in a room temp. glass depends upon suppressing these short-length scale, high-frequency dynamics within the glass. We briefly discuss the coupling of protein motions to the local dynamics of the glass. Also, we show that the glass transition temp. (Tg) alone is not a good indicator for the protein stability in this series of glasses; the glass that confers the max. room-temp. stability does not have the highest Tg.
    52. 52
      Soles, C. L.; Tsai, A. M.; Cicerone, M. T. Misbehaving Proteins: Protein (Mis)Folding, Aggregation, and Stability; Springer New York: New York, 2006; pp 193– 214,  DOI: 10.1007/978-0-387-36063-8_9 .
      There is no corresponding record for this reference.
    53. 53
      Cornicchi, E.; Onori, G.; Paciaroni, A. Picosecond-Time-Scale Fluctuations of Proteins in Glassy Matrices: The Role of Viscosity. Phys. Rev. Lett. 2005, 95, 158104,  DOI: 10.1103/PhysRevLett.95.158104
      53
      Picosecond-Time-Scale Fluctuations of Proteins in Glassy Matrices: The Role of Viscosity
      Cornicchi, Elena; Onori, Giuseppe; Paciaroni, Alessandro
      Physical Review Letters (2005), 95 (15), 158104/1-158104/4CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)
      Through elastic neutron scattering we investigated the fast dynamics of lysozyme in hydrated powder form or embedded in glycerol-water and glucose-water matrixes. We calcd. the relaxational contribution to the mean square displacements of protein hydrogen atoms. We found that the inverse of this quantity is linearly proportional to the logarithm of the viscosity of the solvent glassy matrix. This relationship suggests a close connection between the picosecond-time-scale dynamics of protein side chains and the solvent structural relaxation.
    54. 54
      Wuttke, J.; Budwig, A.; Drochner, M.; Kämmerling, H.; Kayser, F.-J.; Kleines, H.; Ossovyi, V.; Pardo, L. C.; Prager, M.; Richter, D.; Schneider, G. J.; Schneider, H.; Staringer, S. SPHERES, Jülich’s high-flux neutron backscattering spectrometer at FRM II. Rev. Sci. Instrum. 2012, 83, 075109,  DOI: 10.1063/1.4732806
      54
      SPHERES, Juelich's high-flux neutron backscattering spectrometer at FRM II
      Wuttke, Joachim; Budwig, Alfred; Drochner, Matthias; Kaemmerling, Hans; Kayser, Franz-Joseph; Kleines, Harald; Ossovyi, Vladimir; Pardo, Luis Carlos; Prager, Michael; Richter, Dieter; Schneider, Gerald J.; Schneider, Harald; Staringer, Simon
      Review of Scientific Instruments (2012), 83 (7), 075109/1-075109/11CODEN: RSINAK; ISSN:0034-6748. (American Institute of Physics)
      SPHERES is a third-generation neutron backscattering spectrometer, located at the 20 MW German neutron source FRM II and operated by the Juelich Center for Neutron Science. It offers an energy resoln. (fwhm) better than 0.65 μeV, a dynamic range of ± 31 μeV, and a signal-to-noise ratio of up to 1750:1. (c) 2012 American Institute of Physics.
    55. 55
      Zamponi, M.; Khaneft, M. SPHERES: Backscattering spectrometer. J. Large-Scale Res. Facil. 2015, 1, A30,  DOI: 10.17815/jlsrf-1-38
      There is no corresponding record for this reference.
    56. 56
      Natali, F.; Peters, J.; Russo, D.; Barbieri, S.; Chiapponi, C.; Cupane, A.; Deriu, A.; Di Bari, M. T.; Farhi, E.; Gerelli, Y.; Mariani, P.; Paciaroni, A.; Rivasseau, C.; Schiro, G.; Sonvico, F. IN13 Backscattering Spectrometer at ILL: Looking for Motions in Biological Macromolecules and Organisms. Neutron News 2008, 19, 14– 18,  DOI: 10.1080/10448630802474083
      There is no corresponding record for this reference.
    57. 57
      Smith, J. C. Protein dynamics: comparison of simulations with inelastic neutron scattering experiments. Q. Rev. Biophys. 1991, 24, 227– 291,  DOI: 10.1017/S0033583500003723
      57
      Protein dynamics: comparison of simulations with inelastic neutron scattering experiments
      Smith, J. C.
      Quarterly Reviews of Biophysics (1991), 24 (3), 227-91CODEN: QURBAW; ISSN:0033-5835.
      A review with many refs.
    58. 58
      Yi, Z.; Miao, Y.; Baudry, J.; Jain, N.; Smith, J. C. Derivation of Mean-Square Displacements for Protein Dynamics from Elastic Incoherent Neutron Scattering. J. Phys. Chem. B 2012, 116, 5028– 5036,  DOI: 10.1021/jp2102868
      58
      Derivation of Mean-Square Displacements for Protein Dynamics from Elastic Incoherent Neutron Scattering
      Yi, Zheng; Miao, Yinglong; Baudry, Jerome; Jain, Nitin; Smith, Jeremy C.
      Journal of Physical Chemistry B (2012), 116 (16), 5028-5036CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
      The derivation of mean-square displacements from elastic incoherent neutron scattering (EINS) of proteins was examd., with the aid of expts. on camphor-bound cytochrome P450cam and complementary mol. dynamics simulations. A q4 correction to the elastic incoherent structure factor (q is the scattering vector) can be simply used to reliably est. from the expt. both the av. mean-square at. displacement, 〈Δr2〉 of the nonexchanged hydrogen atoms in the protein and its variance, σ2. The mol. dynamics simulation results are in broad agreement with the exptl. derived 〈Δr2〉 and σ2 derived from EINS on instruments at two different energy resolns., corresponding to dynamics on the ∼100 ps and ∼1 ns time scales. Significant dynamical heterogeneity is found to arise from methyl-group rotations. The easy-to-apply q4 correction extends the information extd. from elastic incoherent neutron scattering expts. and should be of wide applicability.
    59. 59
      Parak, F.; Knapp, E. W. A consistent picture of protein dynamics. Proc. Natl. Acad. Sci. U. S. A. 1984, 81, 7088– 7092,  DOI: 10.1073/pnas.81.22.7088
      59
      A consistent picture of protein dynamics
      Parak, F.; Knapp, E. W.
      Proceedings of the National Academy of Sciences of the United States of America (1984), 81 (22), 7088-92CODEN: PNASA6; ISSN:0027-8424.
      Information about the protein dynamics of myoglobin obtained by x-ray and Moessbauer investigations is analyzed and compared with computer simulations. Computer simulations give correct amplitudes of mean-square displacements but fail in the description of the time dependence of motions. A proposed model describes protein dynamics at physiol. temps. as an overdamped diffusion-like motion in a restricted space. The fluctuations occur around the av. conformation detd. by x-ray structure anal. The gain in entropy drives the mol. into the transition state and, in this way, accounts for its flexibility.
    60. 60
      Willis, B.; Pryor, A. Thermal Vibrations in Crystallography; Cambridge University Press, 1975.
      There is no corresponding record for this reference.
    61. 61
      Zeller, D.; Telling, M. T. F.; Zamponi, M.; García-Sakai, V.; Peters, J. Analysis of elastic incoherent neutron scattering data beyond the Gaussian approximation. J. Chem. Phys. 2018, 149, 234908,  DOI: 10.1063/1.5049938
      61
      Analysis of elastic incoherent neutron scattering data beyond the Gaussian approximation
      Zeller, D.; Telling, M. T. F.; Zamponi, M.; Garcia Sakai, V.; Peters, J.
      Journal of Chemical Physics (2018), 149 (23), 234908/1-234908/17CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
      This work addresses the use of the Gaussian approxn. as a common tool to ext. at. motions in proteins from elastic incoherent neutron scattering and whether improvements in data anal. and addnl. information can be obtained when going beyond that. We measured alpha-lactalbumin with different levels of hydration on three neutron backscattering spectrometers, to be able to resolve a wide temporal and spatial range for dynamics. We demonstrate that the Gaussian approxn. gives qual. similar results to models that include heterogeneity, if one respects a certain procedure to treat the intercept of the elastic intensities with the momentum transfer axis. However, the inclusion of motional heterogeneity provides better fits to the data. Our anal. suggests an approach of limited heterogeneity, where including only two kinds of motions appears sufficient to obtain more quant. results for the mean square displacement. Finally, we note that traditional backscattering spectrometers pose a limit on the lowest accessible momentum transfer. We therefore suggest that complementary information about the spatial evolution of the elastic intensity close to zero momentum transfer can be obtained using other neutron methods, in particular, neutron spin-echo together with polarization anal. (c) 2018 American Institute of Physics.
    62. 62
      Frauenfelder, H.; Sligar, S. G.; Wolynes, P. G. The energy landscapes and motions of proteins. Science 1991, 254, 1598– 1603,  DOI: 10.1126/science.1749933
      62
      The energy landscapes and motions of proteins
      Frauenfelder, Hans; Sligar, Stephen G.; Wolynes, Peter G.
      Science (Washington, DC, United States) (1991), 254 (5038), 1598-603CODEN: SCIEAS; ISSN:0036-8075.
      Recent expts., advances in theory, and analogies to other complex systems such as glasses and spin glasses yield insight into protein dynamics. The basis of the understanding is the observation that the energy landscape is complex: proteins can assume a large no. of nearly isoenergetic conformations (conformational substates). The concepts that emerge from studies of the conformational substates and the motions between them permit a quant. discussion of one simple reaction, the binding of small ligands such as carbon monoxide to myoglobin.
    63. 63
      Paciaroni, A.; Cinelli, S.; Cornicchi, E.; Francesco, A. D.; Onori, G. Fast fluctuations in protein powders: The role of hydration. Chem. Phys. Lett. 2005, 410, 400– 403,  DOI: 10.1016/j.cplett.2005.05.098
      63
      Fast fluctuations in protein powders: The role of hydration
      Paciaroni, Alessandro; Cinelli, Stefania; Cornicchi, Elena; De Francesco, Alessio; Onori, Giuseppe
      Chemical Physics Letters (2005), 410 (4-6), 400-403CODEN: CHPLBC; ISSN:0009-2614. (Elsevier B.V.)
      An elastic neutron scattering investigation of the mol. dynamics of hydrated lysozyme powders has been undertaken for different water contents, h (g water/g Lysozyme). The dry sample exhibits a harmonic behavior in the whole temp. range, while anharmonic motions arise on hydrated samples at a temp. T d. Both T d and the magnitude of the anharmonic motions are markedly hydration-dependent. On increasing water content, the crossing barrier entropy change increases, while the enthalpy change keeps const. The estd. av. rigidity of the protein structure decreases abruptly immediately below the onset of the enzymic activation at around 0.2h.
    64. 64
      Schiró, G.; Natali, F.; Cupane, A. Physical Origin of Anharmonic Dynamics in Proteins: New Insights From Resolution-Dependent Neutron Scattering on Homomeric Polypeptides. Phys. Rev. Lett. 2012, 109, 128102,  DOI: 10.1103/PhysRevLett.109.128102
      64
      Physical origin of anharmonic dynamics in proteins: new insights from resolution-dependent neutron scattering on homomeric polypeptides
      Schiro, Giorgio; Natali, Francesca; Cupane, Antonio
      Physical Review Letters (2012), 109 (12), 128102/1-128102/5CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)
      Neutron scattering reveals a complex dynamics in polypeptide chains, with two main onsets of anharmonicity whose phys. origin and biol. role are still debated. In this study the dynamics of strategically selected homomeric polypeptides is investigated with elastic neutron scattering using different energy resolns. and compared with that of a real protein. Our data spotlight the dependence of anharmonic transition temps. and fluctuation amplitudes on energy resoln., which we quant. explain in terms of a two-site model for the protein-hydration water energy landscape. Exptl. data strongly suggest that the protein dynamical transition is not a mere resoln. effect but is due to a real phys. effect. Activation barriers and free energy values obtained for the protein dynamical transition allow us to make a connection with the two-well interaction potential of supercooled-confined water proposed to explain a low-d. → high-d. liq.-liq. transition.
    65. 65
      Stoeckli, A.; Furrer, A.; Schoenenberger, C.; Meier, B.; Ernst, R.; Anderson, I. Dynamics of hydrogen bonds in carboxylic acids. Physica B+C 1986, 136, 161– 164,  DOI: 10.1016/S0378-4363(86)80045-6
      65
      Dynamics of hydrogen bonds in carboxylic acids
      Stoeckli, A.; Furrer, A.; Schoenenberger, C.; Meier, B. H.; Ernst, R. R.; Anderson, I.
      Physica B+C: Physics of Condensed Matter + Atomic, Molecular and Plasma Physics, Optics (Amsterdam) (1986), 136 (1-3), 161-4CODEN: PHBCDQ; ISSN:0378-4363.
      Double proton exchange in cryst. ring-deuterated terephthalic acid and cryst. acetylenedicarboxylic acid was studied by incoherent neutron scattering. The energy spectra above 100 K reveal quasielastic contributions which indicate that the disorder of the H-bonded dimeric units is dynamic involving a translational jump across the hydrogen bonds. The data were analyzed in terms of a two-site jump model.
    66. 66
      Schiró, G. Anharmonic onsets in polypeptides revealed by neutron scattering: Experimental evidences and quantitative description of energy resolution dependence. Biophys. Chem. 2013, 180–181, 29– 36,  DOI: 10.1016/j.bpc.2013.05.006
      66
      Anharmonic onsets in polypeptides revealed by neutron scattering: Experimental evidences and quantitative description of energy resolution dependence
      Schiro, Giorgio
      Biophysical Chemistry (2013), 180-181 (), 29-36CODEN: BICIAZ; ISSN:0301-4622. (Elsevier B.V.)
      Neutron scattering measurements on protein powders reveal 2 deviations from harmonic dynamics at low temp., whose mol. origin, phys. nature, and biol. relevance are still matter of discussion. Here, the author presents a new exptl. and theor. approach to evidence the resoln. dependence of anharmonic onsets: the use of strategically selected homomeric polypeptides, such as polyglycine and polyalanine, allowed revealing the exact resoln. dependence; a 2-site energy landscape model, where resoln. effects are explicitly taken into account, was able to interpret quant. the exptl. data in terms of energy landscape parameters. The energetic description provided by this anal., together with recent exptl. evidences obtained on chem. and structurally different peptide systems, allowed one to connect the protein/water energy landscape structure with the 2-wells water interaction potential proposed to explain the low-d. → high-d. liq.-liq. transition obsd. in supercooled water.
    67. 67
      Afroze, F.; Nies, E.; Berghmans, H. Phase transitions in the system poly(N-isopropylacrylamide)/water and swelling behaviour of the corresponding networks. J. Mol. Struct. 2000, 554, 55– 68,  DOI: 10.1016/S0022-2860(00)00559-7
      67
      Phase transitions in the system poly(N-isopropylacrylamide)/water and swelling behavior of the corresponding networks
      Afroze, F.; Nies, E.; Berghmans, H.
      Journal of Molecular Structure (2000), 554 (1), 55-68CODEN: JMOSB4; ISSN:0022-2860. (Elsevier Science B.V.)
      The phase behavior of linear poly(N-isopropylacrylamide) (PNIPA) and chem. cross-linked PNIPA in water has been detd. by calorimetric and optical techniques. Expts. for the linear polymer were conducted for three different molar masses and encompassed the whole accessible concn. range. In addn. the binodal of the 'monomer' N-(isopropyl)propionamide (NIPPA) was detd. by cloud point measurements. The phase behavior was analyzed in terms of the Flory-Huggins-Staverman theory amended with a strong concn. dependent interaction function. The anal. established that PNIPA/water is exemplar of Type II phase behavior, indicating that the system does not fit the classic Flory-Huggins scheme. Distinctive of Type II behavior is the existence of an off-zero liq.-liq. crit. compn. for a hypothetical polymer of infinite molar wt. and the absence of the classic FH Θ-crit. state. The Type II phase behavior of the linear polymer is also found in the network and is shown to be responsible for the discontinuous vol. response of the PNIPA gel. The peculiar swelling behavior is a direct consequence of the invariant interference of the swelling curve with the liq.-liq. miscibility gap. Finally, the dependence of the enthalpy of melting of water in the polymer soln. is not related to the formation of a strong complex between the polymer and the water mols. Instead, crystn. and melting of water are impeded by the vitrification of the aq. polymer soln.
    68. 68
      Van Durme, K.; Van Assche, G.; Van Mele, B. Kinetics of Demixing and Remixing in Poly(N-isopropylacrylamide)/Water Studied by Modulated Temperature DSC. Macromolecules 2004, 37, 9596– 9605,  DOI: 10.1021/ma048472b
      68
      Kinetics of Demixing and Remixing in Poly(N-isopropylacrylamide)/Water Studied by Modulated Temperature DSC
      Van Durme, Kurt; Van Assche, Guy; Van Mele, Bruno
      Macromolecules (2004), 37 (25), 9596-9605CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
      The heat capacity signal from modulated temp. DSC can be used to measure the onset of phase sepn. in aq. poly(N-isopropylacrylamide) (PNIPAM) solns., showing a type II LCST (lower crit. soln. temp.) demixing behavior. Quasi-isothermal measurements through the phase transition show large excess contributions in the (apparent) heat capacity, caused by demixing and remixing heat effects on the time scale of the modulation. These excess contributions and their time-dependent evolution are useful to describe the kinetics of phase sepn. and to follow the related morphol. development. Partial vitrification of the polymer-rich phase slows down the remixing kinetics.
    69. 69
      Buratti, E.; Tavagnacco, L.; Zanatta, M.; Chiessi, E.; Buoso, S.; Franco, S.; Ruzicka, B.; Angelini, R.; Orecchini, A.; Bertoldo, M.; Zaccarelli, E. The role of polymer structure on water confinement in poly(N-isopropylacrylamide) dispersions. J. Mol. Liq. 2022, 355, 118924,  DOI: 10.1016/j.molliq.2022.118924
      69
      The role of polymer structure on water confinement in poly(N-isopropylacrylamide) dispersions
      Buratti, Elena; Tavagnacco, Letizia; Zanatta, Marco; Chiessi, Ester; Buoso, Sara; Franco, Silvia; Ruzicka, Barbara; Angelini, Roberta; Orecchini, Andrea; Bertoldo, Monica; Zaccarelli, Emanuela
      Journal of Molecular Liquids (2022), 355 (), 118924CODEN: JMLIDT; ISSN:0167-7322. (Elsevier B.V.)
      Poly(N-isopropylacrylamide) (PNIPAM) is a synthetic polymer that is widely studied for its thermoresponsive character. However, recent works also reported evidence of a low temp. (protein-like) dynamical transition around 225 K in concd. PNIPAM suspensions, independently of the polymer architecture, i.e., both for linear chains and for microgels. In this work, we investigate water-polymer interactions by extensive differential scanning calorimetry (DSC) measurements of both systems, in order to understand the effect of the different topol. structures on the soln. behavior, in particular regarding crystn. and melting processes. In addn., we compare protiated and deuterated microgels, in both water and deuterated water. The DSC results are complemented by dynamic light scattering expts., which confirm that the selective isotopic substitution differently affects the soln. behavior. Our findings highlight the important role played by the polymer architecture on the soln. behavior: indeed, microgels turn out to be more efficient confining agents, able to avoid water crystn. in a wider concn. range with respect to linear chains. Altogether, the present data will be valuable to interpret future low-temp. investigations of PNIPAM dispersions, particularly by neutron scattering expts.
    70. 70
      Ping, Z. H.; Nguyen, Q. T.; Chen, S. M.; Zhou, J. Q.; Ding, Y. D. States of water in different hydrophilic polymers ─ DSC and FTIR studies. Polymer 2001, 42, 8461– 8467,  DOI: 10.1016/S0032-3861(01)00358-5
      70
      States of water in different hydrophilic polymers - DSC and FTIR studies
      Ping, Z. H.; Nguyen, Q. T.; Chen, S. M.; Zhou, J. Q.; Ding, Y. D.
      Polymer (2001), 42 (20), 8461-8467CODEN: POLMAG; ISSN:0032-3861. (Elsevier Science Ltd.)
      The structure of water mols. sorbed in different hydrophilic polymers was studied by DSC and FTIR. The obtained data shows that, first, the sorbed water mols. are directly bound to the hydrophilic site to form non-freezable water. Then, beyond a certain water content threshold, the sorbed water mols. become freezable, but with a m.p. lower than 0°C, due to their location in the second hydration layer. Bulk-like water which can be frozen at 0°C appears at higher water contents, and the two types of freezable water finally merge together at very high water contents. The av. no. of non-freezable water mols. per site depends on the chem. nature of the polar site: ca. 1 for a hydroxyl, and 4.2 for an amide group. For a polymer with carboxylate sites, it increases with the size of the alk. counter-ion of the site, due to the increasing ability of the carboxylate counter-ion pair to undergo dissocn.
    71. 71
      Zhang, T.; Li, T.; Nies, E.; Berghmans, H.; Ge, L. Isothermal crystallization study on aqueous solution of poly(vinyl methyl ether) by DSC method. Polymer 2009, 50, 1206– 1213,  DOI: 10.1016/j.polymer.2008.12.038
      71
      Isothermal crystallization study on aqueous solution of poly(vinyl methyl ether) by DSC method
      Zhang, Tianzhu; Li, Ting; Nies, Erik; Berghmans, Hugo; Ge, Liqin
      Polymer (2009), 50 (5), 1206-1213CODEN: POLMAG; ISSN:0032-3861. (Elsevier Ltd.)
      A study on the isothermal crystn. of water in aq. solns. of poly(vinyl Me ether) (PVME) was carried out by the differential scanning calorimetry (DSC). The influence of PVME concn. (49.5, 44.5 and 39.5 v%) and the crystn. temp. (T c) on crystn. rate G, crystn. enthalpy (ΔH c) and melting enthalpy (ΔH m) was investigated. Avrami equation cannot be used to describe the crystn. process of water in aq. PVME soln. Within the measured temp. range, the crystn. rate G increases with the crystn. temp. T c and with the decreasing PVME content. The crystn. enthalpy ΔH c linearly increases with the degree of supercooling. The influence of T c on the ΔH c becomes more marked with increasing PVME concn. For 49.5 and 44.5 v% PVME solns., the amt. of water arrested in soln. during the isothermal crystn. and the final concn. of PVME-rich phase increase linearly with the T c, whereas for 39.5 v% PVME soln., these two values almost do not change with T c. The amt. of frozen water in the subsequent cold crystn. is approx. proportional to the initial T c. The approx. const. ΔH m for a given concn. at the different initial isothermal crystn. temps. suggests that the total amt. of ice from the first isothermal crystn. and the second cold crystn. is same. The quant. relation of the amt. of frozen water in the cold crystn. and the initial T c demonstrates that PVME/water complexes are thermodynamically unstable.
    72. 72
      Tanaka, M.; Motomura, T.; Ishii, N.; Shimura, K.; Onishi, M.; Mochizuki, A.; Hatakeyama, T. Cold crystallization of water in hydrated poly(2-methoxyethyl acrylate) (PMEA). Polym. Int. 2000, 49, 1709– 1713,  DOI: 10.1002/1097-0126(200012)49:12<1709::AID-PI601>3.0.CO;2-L
      72
      Cold crystallization of water in hydrated poly(2-methoxyethyl acrylate) (PMEA)
      Tanaka, Masaru; Motomura, Tadahiro; Ishii, Naoki; Shimura, Kenichi; Onishi, Makoto; Mochizuki, Akira; Hatakeyama, Tatsuko
      Polymer International (2000), 49 (12), 1709-1713CODEN: PLYIEI; ISSN:0959-8103. (John Wiley & Sons Ltd.)
      The structure of water assocd. with poly(2-methoxyethyl acrylate) (PMEA), which is known to exhibit excellent blood compatibility, has been investigated using DSC. The total equil. water content (EWC) of PMEA was 9.0wt%. Water in the PMEA could be classified into three types: non-freezing, freezing-bound and free water. Cold crystn. of water was clearly obsd. at about -42°C on heating when the water content was more than 3.0wt%. Cold crystn. is attributed to the phase transition from the amorphous ice to the crystal ice in PMEA. The relative proportions of freezing-bound water at the EWC is 48% of all the water in hydrated PMEA.
    73. 73
      Guan, L.; Xu, H.; Huang, D. The investigation on states of water in different hydrophilic polymers by DSC and FTIR. Journal of Polymer Research 2011, 18, 681– 689,  DOI: 10.1007/s10965-010-9464-7
      73
      The investigation on states of water in different hydrophilic polymers by DSC and FTIR
      Guan, Lan; Xu, Hongyan; Huang, Dinghai
      Journal of Polymer Research (2011), 18 (4), 681-689CODEN: JPOREP; ISSN:1022-9760. (Springer)
      The interaction between polymers and water in four hydrophilic polymer aq. solns. were investigated by DSC and FTIR. DSC result shows that the different hydrophilic polymer/water mixts. have various water calorimetric behaviors in the melting temp. range of freezable bound water as well as free water. The melting temp. of freezable water and the amt. of non-freezable water in the mixts. vary with the change of chem. structure of polymers. The m.p. of the freezable bound water doesn't change with the water content, revealing that water bound weakly to polymer chains can form a stable cryst. structure at high water content. For the three hydrophilic polymer/water mixts. with C=O group, the wt. ratio of non-freezable water to polymers is const., but varies with polymer chem. structures. The FTIR spectra confirmed the formation of the hydrogen bonds and it was found that there exist different states of water based on various strengths of hydrogen bonds. The OH stretching bands indicated the fraction of strongly bound water decreases with increasing water content. It was concluded that at least in hydrophilic polymer aq. solns. with polar sites in polymer chains, the formation of non-freezable water is ascribed to the hydrogen bonds between hydrophilic polymers and water mols. Different strengths of hydrogen bonds can affect the thermal behaviors of water in the hydrophilic polymer/water mixts.
    74. 74
      Guo, Y.; Sun, B.; Wu, P. Phase Separation of Poly(vinyl methyl ether) Aqueous Solution: A Near-Infrared Spectroscopic Study. Langmuir 2008, 24, 5521– 5526,  DOI: 10.1021/la7038398
      74
      Phase Separation of Poly(vinyl methyl ether) Aqueous Solution: A Near-Infrared Spectroscopic Study
      Guo, Yilu; Sun, Bingjie; Wu, Peiyi
      Langmuir (2008), 24 (10), 5521-5526CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      The thermosensitive phase sepn. of poly(vinyl Me ether) (PVME) aq. solns. was studied using near-IR spectroscopy in combination with two-dimensional correlation anal., and a two-step phase sepn. mechanism during gradual heating was established. Two-dimensional near-IR (2D NIR) anal. results indicate that during this two-step process the dehydration of CH2 groups occurs earlier than that of CH3 groups. This result suggests that it is the change of the hydrophobic hydrocarbon chain conformation induced by heating that indirectly leads to the dehydration of the hydrophilic ether oxygen side groups.
    75. 75
      Nickels, J. D.; O’Neill, H.; Hong, L.; Tyagi, M.; Ehlers, G.; Weiss, K. L.; Zhang, Q.; Yi, Z.; Mamontov, E.; Smith, J. C.; Sokolov, A. P. Dynamics of Protein and its Hydration Water: Neutron Scattering Studies on Fully Deuterated GFP. Biophys. J. 2012, 103, 1566– 1575,  DOI: 10.1016/j.bpj.2012.08.046
      75
      Dynamics of Protein and its Hydration Water: Neutron Scattering Studies on Fully Deuterated GFP
      Nickels, Jonathan D.; O'Neill, Hugh; Hong, Liang; Tyagi, Madhusudan; Ehlers, Georg; Weiss, Kevin L.; Zhang, Qiu; Yi, Zheng; Mamontov, Eugene; Smith, Jeremy C.; Sokolov, Alexei P.
      Biophysical Journal (2012), 103 (7), 1566-1575CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      We present a detailed anal. of the picosecond-to-nanosecond motions of green fluorescent protein (GFP) and its hydration water using neutron scattering spectroscopy and hydrogen/deuterium contrast. The anal. reveals that hydration water suppresses protein motions at lower temps. (<∼200 K), and facilitates protein dynamics at high temps. Exptl. data demonstrate that the hydration water is harmonic at temps. <∼180-190 K and is not affected by the proteins' Me group rotations. The dynamics of the hydration water exhibits changes at ∼180-190 K that we ascribe to the glass transition in the hydrated protein. Our results confirm significant differences in the dynamics of protein and its hydration water at high temps.: on the picosecond-to-nanosecond timescale, the hydration water exhibits diffusive dynamics, while the protein motions are localized to <∼3 Å. The diffusion of the GFP hydration water is similar to the behavior of hydration water previously obsd. for other proteins. Comparison with other globular proteins (e.g., lysozyme) reveals that on the timescale of 1 ns and at equiv. hydration level, GFP dynamics (mean-square displacements and quasielastic intensity) are of much smaller amplitude. Moreover, the suppression of the protein dynamics by the hydration water at low temps. appears to be stronger in GFP than in other globular proteins. We ascribe this observation to the barrellike structure of GFP.
    76. 76
      Sebastiani, F.; Longo, M.; Orecchini, A.; Comez, L.; De Francesco, A.; Muthmann, M.; Teixeira, S. C. M.; Petrillo, C.; Sacchetti, F.; Paciaroni, A. Hydration-dependent dynamics of human telomeric oligonucleotides in the picosecond timescale: A neutron scattering study. J. Chem. Phys. 2015, 143, 015102,  DOI: 10.1063/1.4923213
      76
      Hydration-dependent dynamics of human telomeric oligonucleotides in the picosecond timescale: A neutron scattering study
      Sebastiani, F.; Longo, M.; Orecchini, A.; Comez, L.; De Francesco, A.; Muthmann, M.; Teixeira, S. C. M.; Petrillo, C.; Sacchetti, F.; Paciaroni, A.
      Journal of Chemical Physics (2015), 143 (1), 015102/1-015102/8CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
      The dynamics of the human oligonucleotide, AG3(T2AG3)3, were investigated by incoherent neutron scattering in the sub-nanosecond timescale. A hydration-dependent dynamical activation of thermal fluctuations in weakly hydrated samples was found, similar to that of protein powders. The amplitudes of such thermal fluctuations were evaluated in 2 different exchanged wave-vector ranges, so as to single out the different contributions from intra- and inter-nucleotide dynamics. The activation energy was calcd. from the temp.-dependent characteristic times of the corresponding dynamical processes. The trends of both amplitudes and activation energies support a picture where oligonucleotides possess a larger conformational flexibility than long DNA sequences. This addnl. flexibility, which likely results from a significant relative chain-end contribution to the av. chain dynamics, could be related to the strong structural polymorphism of the investigated oligonucleotides. (c) 2015 American Institute of Physics.
    77. 77
      Russo, D.; Gonzalez, M. A.; Pellegrini, E.; Combet, J.; Ollivier, J.; Teixeira, J. Evidence of Dynamical Constraints Imposed by Water Organization around a Bio–Hydrophobic Interface. J. Phys. Chem. B 2013, 117, 2829– 2836,  DOI: 10.1021/jp3094885
      77
      Evidence of dynamical constraints imposed by water organization around a bio-hydrophobic interface
      Russo, Daniela; Gonzalez, Miguel Angel; Pellegrini, Eric; Combet, J.; Ollivier, J.; Teixeira, Jose
      Journal of Physical Chemistry B (2013), 117 (10), 2829-2836CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
      Mol. dynamics simulations and elastic neutron scattering expts. were used to highlight how the structural organization of hydration water is able in some cases to locally constrain at. movements at biol. interfaces. Using fully hydrated small peptides as models of protein interfaces, the authors show that the length of the side-chains and the hydrophilic backbone have specific signatures. The dynamics of the side-chain, which is part of biomols., play not only a crucial role in the whole flexibility as compared to the backbone, but also modify the values of transition temps. The anal. of the activation energies of Me group dynamics suggests that the interaction between a hydrophobic side-chain and the surrounding water plays an important role in the whole flexibility as well. The authors suggest that the progressive water cluster organization, around hydrophobic interfaces increases the activation energy and that a plateau regime is reached only when an extended H-bond network is established. The cluster size corresponds to a single layer of water mols.
    78. 78
      Hong, L.; Smolin, N.; Lindner, B.; Sokolov, A. P.; Smith, J. C. Three Classes of Motion in the Dynamic Neutron-Scattering Susceptibility of a Globular Protein. Phys. Rev. Lett. 2011, 107, 148102,  DOI: 10.1103/PhysRevLett.107.148102
      78
      Three Classes of Motion in the Dynamic Neutron-Scattering Susceptibility of a Globular Protein
      Hong, Liang; Smolin, Nikolai; Lindner, Benjamin; Sokolov, Alexei P.; Smith, Jeremy C.
      Physical Review Letters (2011), 107 (14), 148102/1-148102/4CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)
      A simplified description of the 295 K dynamics of a globular protein over a wide frequency range (1-1000 GHz) is obtained by combining neutron scattering of lysozyme with mol. dynamics (MD) simulation. The mol. dynamics simulation agrees quant. with expt. for both the protein and the hydration water and shows that whereas the hydration water mols. subdiffuse, the protein atoms undergo confined motion decomposable into three distinct classes: localized diffusion, Me group rotations, and jumps. Each of the three classes gives rise to a characteristic neutron susceptibility signal.
    79. 79
      Schiró, G.; Caronna, C.; Natali, F.; Cupane, A. Direct Evidence of the Amino Acid Side Chain and Backbone Contributions to Protein Anharmonicity. J. Am. Chem. Soc. 2010, 132, 1371– 1376,  DOI: 10.1021/ja908611p
      79
      Direct Evidence of the Amino Acid Side Chain and Backbone Contributions to Protein Anharmonicity
      Schiro, Giorgio; Caronna, Chiara; Natali, Francesca; Cupane, Antonio
      Journal of the American Chemical Society (2010), 132 (4), 1371-1376CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Elastic incoherent neutron scattering has been used to study the temp. dependence of the mean-square displacements of nonexchangeable hydrogen atoms in powders of a series of homomeric polypeptides (polyglycine, polyalanine, polyphenylalanine, and polyisoleucine) in comparison with myoglobin at the same hydration level (h = 0.2). The aim of the work was to measure the dynamic behavior of different amino acid residues sep. and assess the contribution of each type of side chain to the anharmonic dynamics of proteins. The results provide direct exptl. evidence that the first anharmonic activation, at ∼150 K, is largely due to Me group rotations entering the time window of the spectrometer used; however, contributions on the order of 10-20% from the motions of other groups (e.g., the phenolic ring and the methylene groups) are present. Our data also indicate that the dynamical transition occurring at ∼230 K can be attributed, at least at the hydration level investigated, mainly to motions involving backbone fluctuations.
    80. 80
      Schiró, G.; Caronna, C.; Natali, F.; Cupane, A. Molecular origin and hydration dependence of protein anharmonicity: An elastic neutron scattering study. Phys. Chem. Chem. Phys. 2010, 12, 10215– 10220,  DOI: 10.1039/c003482g
      80
      Molecular origin and hydration dependence of protein anharmonicity: An elastic neutron scattering study
      Schiro, Giorgio; Caronna, Chiara; Natali, Francesca; Cupane, Antonio
      Physical Chemistry Chemical Physics (2010), 12 (35), 10215-10220CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)
      Two main onsets of anharmonicity are present in protein dynamics. Neutron scattering on protein hydrated powders revealed a first onset at about 150 K and a second one at about 230 K (the so called dynamical transition). In order to assess the mol. origin of protein anharmonicity, we study different homomeric polypeptides by incoherent elastic neutron scattering, thus disentangling the contribution of different mol. groups in proteins. We show that Me group rotations are the main contributors to the low temp. onset. Concerning the dynamical transition, we show that it also occurs in absence of side chains; however, the presence and mobility of side chains substantially increases the fluctuations amplitude without influencing the transition temp. We also investigate the role of hydration on the anharmonic contributions. Our study shows that Me group rotations are unaffected by hydration and confirms that the dynamical transition is suppressed in dry samples. In hydrated samples, while the pure backbone contribution does not depend on the hydration h at h ≥ 0.2, in the presence of side chains the anharmonic fluctuations involved in the dynamical transition are enhanced by increasing the water content.
    81. 81
      Roh, J. H.; Novikov, V. N.; Gregory, R. B.; Curtis, J. E.; Chowdhuri, Z.; Sokolov, A. P. Onsets of Anharmonicity in Protein Dynamics. Phys. Rev. Lett. 2005, 95, 038101,  DOI: 10.1103/PhysRevLett.95.038101
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      Onsets of Anharmonicity in Protein Dynamics
      Roh, J. H.; Novikov, V. N.; Gregory, R. B.; Curtis, J. E.; Chowdhuri, Z.; Sokolov, A. P.
      Physical Review Letters (2005), 95 (3), 038101/1-038101/4CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)
      Two onsets of anharmonicity are obsd. in the dynamics of the protein lysozyme. One at T∼100 K appears in all samples regardless of hydration level and is consistent with Me group rotation. The second, the well-known dynamical transition at T∼200-230 K, is only obsd. at a hydration level h greater than ∼0.2 and is ascribed to the activation of an addnl. relaxation process. Its variation with hydration correlates well with variations of catalytic activity suggesting that the relaxation process is directly related to the activation of modes required for protein function.
    82. 82
      Frick, B.; Fetters, L. J. Methyl Group Dynamics in Glassy Polyisoprene: A Neutron Backscattering Investigation. Macromolecules 1994, 27, 974– 980,  DOI: 10.1021/ma00082a014
      82
      Methyl Group Dynamics in Glassy Polyisoprene: A Neutron Backscattering Investigation
      Frick, B.; Fetters, L. J.
      Macromolecules (1994), 27 (4), 974-80CODEN: MAMOBX; ISSN:0024-9297.
      Energy-resolved, elastic neutron backscattering was employed to investigate the Me group dynamics in polyisoprene between T = 2 K and room temp. The use of partially deuterated samples and a fully protonated sample allowed the sepn. of the dynamics arising from the Me group and the backbone. An Arrhenius-like increase of the Me group rotational correlation time τ = τ0 exp(Eact/kT), with Eact/k = 1550 K ∼ 12 kJ/mol and Γ0 ∼ 1/τ0 = 23.5 meV (τ0 ∼ 1.76 × 10-13 s) describes the mid-position of the first elastic intensity decrease but not its breadth. A 3-fold jump model with a broad Gaussian distribution of activation energies (dE/E ∼ 25%) around 1500 K can account for the obsd. temp. decrease. Inconstancies in the Q-dependence might be due to disorder effects. The torsional mode of the Me group rotation is directly obsd. at Γ0 = 23.5 meV by time-of-flight. Near the glass transition temp. a further decrease of the elastic scattering is obsd. due to the onset of fast dynamics of the backbone in the picosecond range.
    83. 83
      Rasmussen, D. H.; MacKenzie, A. P. Glass transition in amorphous water. Application of the measurements to problems arising in cryobiology. J. Phys. Chem. 1971, 75, 967– 973,  DOI: 10.1021/j100677a022
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      Glass transition in amorphous water. Application of the measurements to problems arising in cryobiology
      Rasmussen, Don H.; MacKenzie, Alan P.
      Journal of Physical Chemistry (1971), 75 (7), 967-73CODEN: JPCHAX; ISSN:0022-3654.
      Values for the glass transition temp., Tg, of aq. solns. of glycerol, ethylene glycol, and MeOH, measured by DTA, were extrapolated to obtain values for Tg in amorphous water. For a heating rate of 5 deg min-1, Tg for amorphous water is obsd. at -137 ± 1°. The results were correlated with Jenckel's expression for the concn. dependence of Tg in binary solns. A kinetic anal. after McMillan led to "kinetic" parameters for the glass transition that were linear functions of wt. fraction and yielded a time-temp. dependence for Tg in amorphous water correlating well with previously published values.
    84. 84
      Bohon, R.; Conway, W. DTA studies on the glycerol─water system. Thermochim. Acta 1972, 4, 321– 341,  DOI: 10.1016/0040-6031(72)87016-3
      84
      DTA studies on the glycerol-water system
      Bohon, R. L.; Conway, W. T.
      Thermochimica Acta (1972), 4 (3), 321-41CODEN: THACAS; ISSN:0040-6031.
      A modified duPont 900 DTA cell was used to study the complex phase transitions in the glycerol-H2O system at relatively rapid rates of heating and cooling and under carefully monitored thermal history conditions. This system is of particular interest in the cryoprotection of red blood cells. The separation of crystals and a const.-compn. amorphous phase from H2O-rich systems tends to distort the glass transition (Tg) vs. compn. curves unless special precautions are exercised. Extrapolation of Tg to zero glycerol content gave a hypothetical 2nd-order transition in "amorphous" ice of -123 ± 1° at a heating rate of 30°/min. The glycerol-H2O system may exhibit the phenomena of vitreous polymorphs wherein 2 distinct amorphous phases coexist in metastable equil. in the solid state. The obsd. 2nd-order transition in 1 of these phases occurred near, and in some cases slightly above, Tg for pure glycerol. Limited low temp. x-ray diffraction studies on 43.7% glycerol showed the existence of 2 crystalline forms, 1 of which could only be formed by rapid cooling from the liq. phase. Neither form gave diffraction patterns corresponding to known forms of ice.
    85. 85
      Hatakeyama, H.; Yoshida, H.; Nakano, J. Studies on the isothermal crystallization of D-glucose and cellulose oligosaccharides by differential scanning calorimetry. Carbohydr. Res. 1976, 47, 203– 211,  DOI: 10.1016/S0008-6215(00)84185-3
      85
      Studies on the isothermal crystallization of D-glucose and cellulose oligosaccharides by differential scanning calorimetry
      Hatakeyama, Hyoe; Yoshida, Hiroshisa; Nakano, Junzo
      Carbohydrate Research (1976), 47 (2), 203-11CODEN: CRBRAT; ISSN:0008-6215.
      Isothermal crystn. from the glassy state of D-glucose and cellulose oligosaccharides (e.g., cellobiose, cellotriose, and cellotetraose) was studied by differential scanning calorimetry. The crystn. of amorphous D-glucose and oligosaccharides was very difficult in the absence of traces of water. Amorphous cellobiose and cellotetraose crystd. far more rapidly than amorphous D-glucose and cellotriose. The activation energy for the crystn. of cellobiose and cellotetraose was ∼10-12 kJ-mole-1, while that for D-glucose and cellotriose was ∼1-2 kJ-mole-1. An odd-even effect seemed to be assocd. with the crystn. process of these saccharides.
    86. 86
      Noel, T. R.; Parker, R.; Ring, S. G. A comparative study of the dielectric relaxation behaviour of glucose, maltose, and their mixtures with water in the liquid and glassy states. Carbohydr. Res. 1996, 282, 193– 206,  DOI: 10.1016/0008-6215(95)00388-6
      86
      A comparative study of the dielectric relaxation behavior of glucose, maltose, and their mixtures with water in the liquid and glassy states
      Noel, Timothy R.; Parker, Roger; Ring, Stephen G.
      Carbohydrate Research (1996), 282 (2), 193-206CODEN: CRBRAT; ISSN:0008-6215. (Elsevier)
      The dielec. relaxation behavior of glucose, maltose, and their mixts. with water up to a concn. of 12.0 and 23.0% wt./wt., resp., were examd. in the frequency range 102 to 105 Hz. A primary relaxation was obsd. at temps. above the glass transition temp., Tg, and a secondary relaxation at sub-Tg temps. The addn. of water shifted the primary relaxations to lower temps. For the glucose mixts., water increased the strength of the secondary relaxation and resulted in a merging of the primary and secondary relaxations. The increase in strength of the secondary relaxation was much more marked for the maltose-water mixts. and, in this case, the relaxations remained sep. over the range of frequency and water contents studied. For the maltose-water mixts., the dependence of the strength of the secondary relaxation on the water content was bilinear with a change in gradient at ∼10.0% wt./wt. water. The sub-Tg relaxations were thought to arise from motions of pendant hydroxymethyl groups attached to the hexose rings and from the reorientation of water mols. The difference in the secondary relaxation behavior of glucose and maltose indicates that structural factors, in addn. to the presence of hydroxymethyl groups, are also important.
    87. 87
      Shpigelman, A.; Portnaya, I.; Ramon, O.; Livney, Y. D. Saccharide-structure effects on poly N-isopropylacrylamide phase transition in aqueous media; Reflections on protein stability. J. Polym. Sci., Part B: Polym. Phys. 2008, 46, 2307– 2318,  DOI: 10.1002/polb.21562
      87
      Saccharide-structure effects on poly N-isopropylacrylamide phase transition in aqueous media; reflections on protein stability
      Shpigelman, Avi; Portnaya, Irina; Ramon, Ory; Livney, Yoav D.
      Journal of Polymer Science, Part B: Polymer Physics (2008), 46 (21), 2307-2318CODEN: JPBPEM; ISSN:0887-6266. (John Wiley & Sons, Inc.)
      Protein stability in aq. solns. is important in numerous fields, particularly biotechnol. and food-science. To shed new light on the protective effect of carbohydrates on proteins, we studied saccharide-structure effects in aq. solns. on the coil-to-globule transition occurring at the lower crit. soln. temp. (LCST) of poly-N-isopropylacrylamide (PNIPA), an isomer of polyleucine, as a simple model representing certain key behaviors of proteins (e.g., denaturation/renaturation). We systematically selected sugars and polyols to relate structural and phys. characteristics of these carbohydrates to their effect on PNIPA solns. Using isothermal titrn.-microcalorimetry, we showed that no significant binding of saccharides to the polymer occurs. Using micro-DSC, we studied the decreasing polymer LCST temp. with rising carbohydrate concn. Beyond the expected observation that steric exclusion is important, we obsd. previously-unreported significant differences among the effects of isomeric aldohexoses and also among the effects of isomeric diglucoses on PNIPA LCST. We found good correlation between the sugar hydration no. and its effect on LCST. We conclude that the larger and denser the hydrated cluster a carbohydrate forms, the worse a cosolvent is for the polymer, and the stronger it's lowering effect of the coil-to-globule transition. Such favoring of the compact globule state provides a protective effect against denaturation of globular proteins.
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      Corezzi, S.; Bracco, B.; Sassi, P.; Paolantoni, M.; Comez, L. Protein Hydration in a Bioprotecting Mixture. Life 2021, 11, 995,  DOI: 10.3390/life11100995
      88
      Protein Hydration in a Bioprotecting Mixture
      Corezzi, Silvia; Bracco, Brenda; Sassi, Paola; Paolantoni, Marco; Comez, Lucia
      Life (Basel, Switzerland) (2021), 11 (10), 995CODEN: LBSIB7; ISSN:2075-1729. (MDPI AG)
      We combined broad-band depolarized light scattering and IR spectroscopies to study the properties of hydration water in a lysozyme-trehalose aq. soln., where trehalose is present above the concn. threshold (30% in wt.) relevant for biopreservation. The joint use of the two different techniques, which were sensitive to inter-and intra-mol. degrees of freedom, shed new light on the mol. mechanism underlying the interaction between the three species in the mixt. Thanks to the comparison with the binary soln. cases, we were able to show that, under the investigated conditions, the protein, through preferential hydration, remains strongly hydrated even in the ternary mixt. This supported the water entrapment scenario, for which a certain amt. of water between protein and sugar protects the biomol. from damage caused by external agents.
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      Shimizu, S.; Smith, D. J. Preferential hydration and the exclusion of cosolvents from protein surfaces. J. Chem. Phys. 2004, 121, 1148– 1154,  DOI: 10.1063/1.1759615
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      Preferential hydration and the exclusion of cosolvents from protein surfaces
      Shimizu, Seishi; Smith, Derek J.
      Journal of Chemical Physics (2004), 121 (2), 1148-1154CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
      Protein stability is enhanced by the addn. of osmolytes, such as sugars and polyols and inert crowders, such as polyethylene glycols. This stability enhancement has been quantified by the preferential hydration parameter which can be detd. by expts. To understand the mechanism of protein stability enhancement, we present a statistical mech. anal. of the preferential hydration parameter based upon Kirkwood-Buff theory. Previously, the preferential hydration parameter was interpreted in terms of the no. of hydration waters, as well as the cosolvent exclusion vol. It was not clear how accurate these interpretations were, nor what the relationship is between the two. By using the Kirkwood-Buff theory and exptl. data, we conclude that the contribution from the cosolvent exclusion dominantly dets. the preferential hydration parameters for crowders. For osmolytes, although the cosolvent exclusion largely dets. the preferential hydration parameters, the contribution from hydration may not be negligible.
    90. 90
      Arakawa, T.; Timasheff, S. N. Stabilization of protein structure by sugars. Biochemistry 1982, 21, 6536– 6544,  DOI: 10.1021/bi00268a033
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      Stabilization of protein structure by sugars
      Arakawa, Tsutomu; Timasheff, Serge N.
      Biochemistry (1982), 21 (25), 6536-44CODEN: BICHAW; ISSN:0006-2960.
      The preferential interaction of proteins with solvent components was measured in aq. lactose and glucose systems by using a high precision densimeter. In all cases, the protein was preferentially hydrated; i.e., addn. of these sugars to an aq. soln. of the protein resulted in an unfavorable free-energy change. This effect increased with an increase in protein surface area, explaining the protein-stabilizing action of these sugars and their enhancing effect of protein assocns. Correlation of the preferential interaction parameter with the effect of the sugars on the surface tension of water, i.e., their pos. surface tension increment, led to the conclusion that the surface free energy perturbation by sugars plays a predominant role in their preferential interaction with proteins. Other contributing factors were the exclusion vol. of the sugars and the chem. nature of the protein surface.
    91. 91
      Zaccai, G. How Soft Is a Protein? A Protein Dynamics Force Constant Measured by Neutron Scattering. Science 2000, 288, 1604– 1607,  DOI: 10.1126/science.288.5471.1604
      91
      How soft is a protein? A protein dynamics force constant measured by neutron scattering
      Zaccai, Giuseppe
      Science (Washington, D. C.) (2000), 288 (5471), 1604-1607CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      A review with 32 refs. An effective environmental force const. is introduced to quantify the mol. resilience (or its opposite, "softness") of a protein structure and relate it to biol. function and activity. Specific resilience-function relations were found in neutron-scattering expts. on purple membranes contg. bacteriorhodopsin, the light-activated proton pump of halobacteria; the connection between resilience and stability is illustrated by a study of myoglobin in different environments. Important advantages of the neutron method are that it can characterize the dynamics of any type of biol. sample, which need not be cryst. or monodisperse, and that it enables researchers to focus on the dynamics of specific parts of a complex structure with deuterium labeling.
    92. 92
      Gottfried, D. S.; Peterson, E. S.; Sheikh, A. G.; Wang, J.; Yang, M.; Friedman, J. M. Evidence for Damped Hemoglobin Dynamics in a Room Temperature Trehalose Glass. J. Phys. Chem. 1996, 100, 12034– 12042,  DOI: 10.1021/jp9609489
      92
      Evidence for Damped Hemoglobin Dynamics in a Room Temperature Trehalose Glass
      Gottfried, David S.; Peterson, Eric S.; Sheikh, Asim G.; Wang, Jiaqian; Yang, Ming; Friedman, Joel M.
      Journal of Physical Chemistry (1996), 100 (29), 12034-12042CODEN: JPCHAX; ISSN:0022-3654. (American Chemical Society)
      Upon photodissocn. of its ligand, COHbA exhibits a wide range of nonequil. relaxation phenomena that start within a fraction of a picosecond and extend out to tens of microseconds. In addn., equil. fluctuations of the protein result in conformational averaging. All of these dynamics can have an impact on ligand rebinding. In an effort to better understand the relation between conformational dynamics and ligand-binding reactivity, COHbA was embedded in a room temp. trehalose sugar glass (Hagen et al. Science 1995, 269, 959) to uncouple solvent motions from protein dynamics as well as reduce the amplitude of large-scale protein conformational fluctuations. Time-resolved resonance Raman spectroscopy and ligand-rebinding kinetics show that the trehalose glass does not impede the initial fast relaxation of the iron-histidine linkage, but does dramatically impede conformational averaging and completely eliminates ligand escape at all temps. from 140 K to room temp. Fluorescence measurements indicate that in the trehalose glass the picosecond tryptophan lifetimes are nearly unchanged, but there is a complete absence of the nanosecond fluorescence decay (obsd. in aq. solns.), which is replaced by a decay of ∼700 ps. This change in the fluorescence decay is ascribed to a significant decrease in the structural dynamics that normally allow transient opening of the distal heme pocket.
    93. 93
      Cicerone, M. T.; Douglas, J. F. β-Relaxation governs protein stability in sugar-glass matrices. Soft Matter 2012, 8, 2983– 2991,  DOI: 10.1039/c2sm06979b
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      β-Relaxation governs protein stability in sugar-glass matrices
      Cicerone, Marcus T.; Douglas, Jack F.
      Soft Matter (2012), 8 (10), 2983-2991CODEN: SMOABF; ISSN:1744-683X. (Royal Society of Chemistry)
      The stabilizing effect of sugar-glass matrix materials for freeze-drying proteins or nucleic acids has been variously ascribed to the thermodn. effect of water replacement by sugar mols. or to the kinetic effect of slowed α relaxation assocd. with sugar matrix vitrification. While evidence for each of these hypotheses exists, neither can adequately account for the obsd. stabilization of proteins embedded in sugar-glasses. Instead, firm evidence that protein stability in these glasses is directly linked to high frequency β relaxation processes of the sugar matrix were found. Specifically, when the β relaxation time, τβ, of sugar-glasses is increased with antiplasticizing additives, protein stability increases in linear proportion to the increase in τβ, even though these same additives simultaneously decrease the glass transition temp., Tg, and the α relaxation time, τα, of the sugar matrix materials. Moreover, while sugars replace water by stabilizing protein native-like conformation in the dry state, the resulting enhanced protein conformational stability does not have a significant impact on the degrdn. rate of the proteins in sugar-glasses. The authors discuss the implications of these findings for the fundamental physics of glass formation and for effective engineering of protein stabilizing glasses through the modification of τβ.
    94. 94
      Lerbret, A.; Affouard, F. Molecular Packing, Hydrogen Bonding, and Fast Dynamics in Lysozyme/Trehalose/Glycerol and Trehalose/Glycerol Glasses at Low Hydration. J. Phys. Chem. B 2017, 121, 9437– 9451,  DOI: 10.1021/acs.jpcb.7b07082
      94
      Molecular Packing, Hydrogen Bonding, and Fast Dynamics in Lysozyme/Trehalose/Glycerol and Trehalose/Glycerol Glasses at Low Hydration
      Lerbret, Adrien; Affouard, Frederic
      Journal of Physical Chemistry B (2017), 121 (40), 9437-9451CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
      Water and glycerol are well-known to facilitate the structural relaxation of amorphous protein matrixes. However, several studies evidenced that they may also limit fast (approx. picosecond-nanosecond) and small-amplitude (approx. Angstrom) motions of proteins, which govern their stability in freeze-dried sugar mixts. To det. how they interact with proteins and sugars in glassy matrixes and, thereby, modulate their fast dynamics, we performed mol. dynamics (MD) simulations of lysozyme/trehalose/glycerol (LTG) and trehalose/glycerol (TG) mixts. at low glycerol and water concns. Upon addn. of glycerol and/or water, the glass transition temp. (Tg) of LTG and TG mixts. decreased, the mol. packing of glasses was improved, and the mean-square displacements (MSDs) of lysozyme and trehalose either decreased or increased, depending on the time scale and on the temp. considered. A detailed anal. of the H-bonds (HBs) formed between species revealed that water and glycerol may antiplasticize the fast dynamics of lysozyme and trehalose by increasing the total no. and/or the strength of the HBs they form in glassy matrixes.
    95. 95
      García-Sakai, V.; Khodadadi, S.; Cicerone, M. T.; Curtis, J. E.; Sokolov, A. P.; Roh, J. H. Solvent effects on protein fast dynamics: implications for biopreservation. Soft Matter 2013, 9, 5336– 5340,  DOI: 10.1039/c3sm50492a
      There is no corresponding record for this reference.

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