Site-Specific Internal Motions in GB1 Protein Microcrystals Revealed by 3D 2H–13C–13C Solid-State NMR Spectroscopy
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Department of Chemistry, University of Illinois at Urbana−Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, United States
*E-mail: [email protected]
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Abstract
2H quadrupolar line shapes deliver rich information about protein dynamics. A newly designed 3D 2H–13C–13C solid-state NMR magic angle spinning (MAS) experiment is presented and demonstrated on the microcrystalline β1 immunoglobulin binding domain of protein G (GB1). The implementation of 2H–13C adiabatic rotor-echo-short-pulse-irradiation cross-polarization (RESPIRATION CP) ensures the accuracy of the extracted line shapes and provides enhanced sensitivity relative to conventional CP methods. The 3D 2H–13C–13C spectrum reveals 2H line shapes for 140 resolved aliphatic deuterium sites. Motional-averaged 2H quadrupolar parameters obtained from the line-shape fitting identify side-chain motions. Restricted side-chain dynamics are observed for a number of polar residues including K13, D22, E27, K31, D36, N37, D46, D47, K50, and E56, which we attribute to the effects of salt bridges and hydrogen bonds. In contrast, we observe significantly enhanced side-chain flexibility for Q2, K4, K10, E15, E19, N35, N40, and E42, due to solvent exposure and low packing density. T11, T16, and T17 side chains exhibit motions with larger amplitudes than other Thr residues due to solvent interactions. The side chains of L5, V54, and V29 are highly rigid because they are packed in the core of the protein. High correlations were demonstrated between GB1 side-chain dynamics and its biological function. Large-amplitude side-chain motions are observed for regions contacting and interacting with immunoglobulin G (IgG). In contrast, rigid side chains are primarily found for residues in the structural core of the protein that are absent from protein binding and interactions.
Introduction
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Protein dynamics plays critical roles in biological functions such as enzyme catalysis, ligand binding, and signal transduction. (1, 2) It has drawn increasing research attention in recent studies, as protein structures alone do not fully explain biological activities. X-ray diffraction (XRD) and nuclear magnetic resonance (NMR) are the premier methods to determine protein conformation. XRD reports primarily on structure, and the protein flexibility is only indirectly reflected in B-factors. Therefore, it is problematic to address protein dynamics solely from B-factors, which also are affected by crystal packing defects, whole-body motions and refinement artifacts. (3, 4) Further, B-factors are insensitive to reorientation motions and are unable to distinguish motions covering different time scales. (3, 4) Protein dynamics can be elucidated by NMR (5-11) along with several other techniques, including MD simulations, (12, 13) fluorescence spectroscopy, (14, 15) and electron paramagnetic resonance (EPR). (16, 17) NMR is the premier technique for probing motions in proteins and widely implemented to investigate protein mobility at the atomic level. (5, 7, 11) Well-developed methods in solution-state NMR include spin relaxation (R1, R2, and R1ρ), (18-21) Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion, (22-24) residual dipolar couplings (RDCs), (25, 26) hydrogen–deuterium exchange, (27) and deuterium relaxation. (28) These measurements capture motions with time scales from picoseconds to seconds, (10, 11, 29-32) which substantially expand our understanding of protein structure and dynamics. For example, a recent study successfully quantified side-chain χ1 distributions by RDCs for the third domain of protein G. (33) Slow molecular tumbling leads to poor spectral resolution and sensitivity, which hinder the application of solution-state NMR to large proteins. In addition, chemical shift anisotropy (CSA), dipolar coupling, and quadrupolar coupling line shapes convey rich information on site-specific motional modes and rates. (7, 34-36) These anisotropic interactions are extensively averaged by isotropic Brownian tumbling motions in solution. In contrast, in solid-state NMR, the anisotropic line shapes are retrieved and can be detected accurately to illustrate dynamics of chemical groups in a protein. Similar to solution-state NMR, spin relaxation is routinely implemented in solid-state NMR for investigating dynamics. (7, 8, 37-40) For example, a recent study determined 13 sets of bulk NMR relaxation times for the β1 immunoglobulin binding domain of protein G (GB1) microcrystals at various temperatures. (41) The results explored the hierarchical distribution of backbone and side-chain motions as well as protein–solvent motion coupling for microcrystalline GB1 over the temperature window of 105–280 K. Recent studies showed that the CPMG relaxation dispersion approach was applicable in the solid state. (42) These solid-state NMR developments paved the way for the elucidation of dynamics in large proteins and especially benefit the study of membrane proteins. Further, high similarity was shown for picosecond to submicrosecond dynamics elucidated by solid-state and solution-state NMR for SH3 and ubiquitin, implying the validation of extrapolating solid-state dynamics information to proteins in their native states. (43-45)
Extensive effort has been devoted to develop solid-state NMR pulse sequences for dynamics detection by utilizing dipole–dipole, CSA, and quadrupolar interactions. For example, studies managed to determine order parameters from scaled 1H–13C dipolar couplings measured using homonuclear coupling attenuation pulse schemes such as T-MREV, (46) phase-modulated Lee–Goldberg irradiation, (47) rotational-echo double resonance (REDOR), (48-51) cross polarization combined with phase inversion (CPPI), (52, 53) and symmetry-based pulse sequences. (54) Moreover, the use of these dipolar recoupling techniques in solid-state NMR has enabled the elucidation of dynamics for a number of proteins. (55-60) The most commonly used quadrupolar nuclei in biological studies is deuterium, which can be enriched by replacing protons without altering critical chemical and physical properties of the systems. In comparison with the dipolar interaction and CSA, the 2H quadrupolar coupling constant (CQ) is significantly larger (∼200 kHz), which gives rise to opportunities to study dynamics as well as several spectroscopic challenges. The large amplitude of the 2H CQ enables much more accurate measurements than those of the dipolar coupling or CSA. (61-65) Currently, however, the ability to quantitatively evaluate protein dynamics is partially complicated by the absence of a complete understanding of rigid-limit quadrupolar tensor values (addressed in the following section). 2H line shape and relaxation time together enable the characterization of protein dynamics covering time scales from picoseconds to seconds. (66-68) On the other hand, limitations of NMR hardware and pulse sequences impede the application of 2H CQ measurements in large proteins or other macromolecules. Solid-state NMR probes enabling high power irradiation of 2H, simultaneously with 1H and 13C and/or 15N, combined with high magic angle spinning (MAS) rates, allow the efficient excitation and detection of 2H signals in a site-specific fashion with multidimensional NMR. Cross-polarization (CP) (69, 70) is one of the most common pulse elements exploited to transfer magnetization in multidimensional solid-state NMR. Several recent studies implemented 2H–13C tangent CP transfers in two-/three-dimensional (2D/3D) experiments to indirectly detect 2H quadrupolar line shapes and spin–lattice (T1) relaxation times to probe dynamics of NAV, (71) amino acids, (72) SH3 protein, (73) and silk proteins. (74, 75) As pointed out in previous studies and observed in the current work, conventional 2H–13C CP schemes lead to nonuniform magnetization transfer across the broad 2H powder pattern. (72) The optimal CP condition providing accurate 2H line shape covers an extraordinarily narrow rf band (<1 kHz) and is sensitive to 2H CQ. (72) Thus, it is challenging to measure quadrupolar parameters accurately for sites with different CQ values using one particular CP condition. This impedes the utilization of conventional 2H–13C CP in multidimensional NMR to extract 2H quadrupolar information for proteins as the range of motional averaged CQ is up to ∼185 kHz.
A recently invented polarization transfer pulse scheme, rotor-echo-short-pulse-irradiation (RESPIRATION) CP, (76-79) shows the potential to overcome the nonuniformity of magnetization transfer observed in conventional CP. This approach provides significantly enhanced efficiency for 13C–15N, 1H–15N, as well as 2H–13C CP and is more tolerant to the variation of experimental conditions such as rf mismatching, probe detuning, and spinning instability. (78) Later studies demonstrated that adiabatic RESPIRATION CP further improves the performance even using very low rf field strengths. (77, 79) These studies primarily focused on the CP efficiency enhancement illustrated by numerical simulations and experimental data. The observed broad 2H–13C CP matching profile in adiabatic RESPIRATION CP implies that an optimal CP condition concurrently satisfies chemical groups with different CQ’s, namely, allowing accurate measurement of quadrupolar coupling parameters for all sites in complicated systems like perdeuterated proteins.
In the present work, the adiabatic RESPIRATION CP element is implemented in a 3D 2H–13C–13C solid-state NMR experiment to elucidate site-specific protein backbone and side-chain dynamics. We show with crystalline Ala that superior 2H–13C magnetization transfer uniformity is observed using adiabatic RESPIRATION CP transfer. SPC-5 (80, 81) is employed in the 3D experiment to build 13C–13C correlations in order to resolve 2H sites. In comparison with commonly used multidimensional experiments that measure 1H–13C/15N dipolar coupling constants, the presented 3D 2H–13C–13C approach has several advantages for dynamics detection. First, the resulting 2H powder patterns are on the order of 100 kHz, which is significantly larger than 1H–13C/15N dipolar spectra covering less than ∼23 kHz. Thus, the current 3D 2H–13C–13C method is less prone to measurement error. Second, dipolar measurements have higher demands on experimental conditions including spinning rates and recoupling pulses in order to efficiently suppress homonuclear dipolar couplings and reintroduce heteronuclear dipolar couplings under MAS. In contrast, the 3D 2H–13C–13C experiment is more robust because the accuracy of the obtained 2H spectra solely depends on the 2H–13C CP step and is ensured by the use of the adiabatic RESPIRATION CP. The third benefit of the 3D 2H–13C–13C approach is that it offers improved spectral resolution through using samples which are typically fully deuterated, relative to dipolar measurements requiring protonated samples. However, one drawback to 2H NMR is that the current understanding of rigid-limit CQ values for proteins is incomplete, consequently impeding the ability to access quantitative dynamics information. Specifically, due to a general lack of systematic studies, the variation of quadrupolar rigid-limit CQ values for deuterium bonded to sp3-hybridized carbon has not been fully evaluated for proteins. To assist quantification of protein side-chain motions using 2H relaxation times, two recent solution-state NMR studies indirectly determined the rigid-limit CQ values for CD3 in the N-terminal drk SH3 domain (82) as well as CαDα in both ubiquitin and GB1 proteins. (83) The methyl deuterium rigid-limit CQ values were found to be approximately uniform, at 167 ± 1.5 kHz. (82)2Hα rigid-limit CQ values were determined to be 174 kHz on average with 6–8% uncertainty which the authors attribute to possible measurement uncertainties rather than actual CQ variation. (83) Together, these two studies suggest that the rigid-limit CQ values for deuterium at methyl and Cα sites in a protein are likely uniform. It is worth noting that the quantification of rigid-limit CQ values in these studies may be associated with nontrivial uncertainties resulting from a number of factors. (82, 83) To date, the rigid-limit CQ values for deuterium at sites other than methyl and Cα sites have not been systematically evaluated for proteins. The inherent variation of deuterium quadrupolar interactions in the rigid lattice requires further investigation in order to guide the 2H solution-state and solid-state NMR dynamics studies for proteins. For example, one straightforward approach to obtain the rigid-limit CQ values is by the measurement of 2H line shapes for proteins with multidimensional MAS solid-state NMR at ultralow temperatures where the motions are quenched, which can be performed with the state-of-the-art NMR instrumentation and is an interesting topic for future studies. In this paper, we focus on the comparison of motionally averaged quadrupolar coupling constant (
) values for side chains of microcrystalline protein GB1 to gain dynamics information. Because of the lack of the quantitative rigid-limit CQ values and the complexity brought by the nonzero motional averaged asymmetry parameters (η̅), we interpret the data by comparing 
values instead of using generalized order parameters. Such comparisons are performed between the same chemical groups of the same type of residues to minimize uncertainties originating from the potential rigid-limit CQ differences. This semiquantitative analysis provides critical insights into protein dynamics, especially those of side chains, which have not been systematically studied in detail.
) values for side chains of microcrystalline protein GB1 to gain dynamics information. Because of the lack of the quantitative rigid-limit CQ values and the complexity brought by the nonzero motional averaged asymmetry parameters (η̅), we interpret the data by comparing values instead of using generalized order parameters. Such comparisons are performed between the same chemical groups of the same type of residues to minimize uncertainties originating from the potential rigid-limit CQ differences. This semiquantitative analysis provides critical insights into protein dynamics, especially those of side chains, which have not been systematically studied in detail.In this study, we demonstrate the 3D 2H–13C–13C experiment on microcrystalline GB1, in order to extract 2H quadrupolar information for each chemical group. The obtained 
and η̅ elucidate the backbone and side-chain motions for the majority of residues in a site-specific manner. To our knowledge, this is the first example in which the backbone and side-chain dynamic network is fully mapped based on 2H quadrupolar coupling parameters extracted from a single solid-state NMR experiment. The results illustrate that side-chain dynamics of GB1 highly correlate with its structure stability and biological functions. We envisage that this approach will be widely applicable to investigate backbone and side-chain motions in various biological systems such as protein microcrystals/nanocrystals, insoluble fibrils, and membrane proteins.
and η̅ elucidate the backbone and side-chain motions for the majority of residues in a site-specific manner. To our knowledge, this is the first example in which the backbone and side-chain dynamic network is fully mapped based on 2H quadrupolar coupling parameters extracted from a single solid-state NMR experiment. The results illustrate that side-chain dynamics of GB1 highly correlate with its structure stability and biological functions. We envisage that this approach will be widely applicable to investigate backbone and side-chain motions in various biological systems such as protein microcrystals/nanocrystals, insoluble fibrils, and membrane proteins.Materials and Methods
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Preparation of Protein GB1
Uniformly labeled 2H,13C,15N-GB1 was expressed in E. coli BL12(DE3) using a previously published protocol. (84) The protein solution was buffer exchanged against 90/10 D2O/H2O to replace 10% of the exchangeable deuterons with protons and then was concentrated to 25 mg/mL and precipitated with 3.0 equiv of 2-methyl-2,4-pentanediol (MPD) and isopropanol (IPA) solution (2:1 MPD/IPA volume ratio). (84) Microcrystalline GB1 was packed into a 1.6 mm standard wall FastMAS rotor (Agilent Technologies Inc.) for use in solid-state NMR experiments.
Solid-State NMR Experiments
Data were collected on a customized 500 MHz Varian VNMRS DirectDrive spectrometer equipped with actively biased transmit–receive circuits and a 1.6 mm FastMAS quadruple resonance 1H–13C–2H–15N probe. A 22.222 kHz MAS was chosen as a compromise between peak sensitivity and the number of sidebands present from the 2H manifold. The variable temperature was 0 °C, and the actual sample temperature was 3 °C because of frictional heating induced by MAS (as determined by an ethylene glycol calibration (85)). The 3D 2H–13C–13C solid-state NMR pulse sequence is displayed in Figure 1A and will be discussed in detail in the following section. The 1H, 13C, 2H, and 15N π/2 pulse widths were 1.5, 1.6, 2.9, and 4.5 μs, respectively. In experiments performed on GB1, 86 kHz 2H and 100 kHz 13C spin-lock rf field strengths and a 900 μs contact time were used for adiabatic RESPIRATION CP magnetization transfer. The rotor-synchronized adiabatic RESPIRATION CP waveform was defined by the following optimized parameters: Δ = 4000 rad/s and b = 5000/2π Hz and RESPIRATION pulse τp = 1.8 μs (see Jain et al. for parameter definition (79)). The SPC-5 homonuclear recoupling scheme (80, 81) was implemented to build 13C–13C correlations with a 1.086 ms mixing time. Additional experimental parameters include a 100 ms recycle delay, 400 kHz 2H sweep width, 22.222 kHz and 50 kHz 13C sweep width for the second and third dimension, 192 t1 increment points, 160 t2 increment points, and a 20.48 ms acquisition time. Low-power 1H XiX (86) and 15N WALTZ (87) decoupling was employed during the pulse periods as indicated in Figure 1A. Data were processed in nmrPipe (88) and analyzed in Sparky (T. D. Goddard and D. G. Kneller, University of California, San Francisco). 2H MAS line shapes were extracted from the 3D 2H–13C–13C correlation experiment and fitted in DMFit. (89)
Results and Discussion
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2H–13C Adiabatic RESPIRATION CP Enabling Accurate Indirect Detection of 2H Line Shapes
Figure 1A shows the 3D 2H–13C–13C NMR pulse sequence that was used for detecting 2H quadrupolar line shapes. The initial excited 2H magnetization was transferred to the directly bonded 13C and detected through 13C–13C correlation. One critical element is the CP transfer that dictates the accuracy of the extracted 2H line shapes and the sensitivity of the experiment. Conventional tangent CP was previously employed in 2D 2H–13C experiments to establish heteronuclear correlation in order to extract 2H line shapes for peptides and proteins. (71-75) One drawback of the approach is that polarization transfer efficiency is not uniform across 2H MAS manifolds. (72) Thus, an optimal CP condition providing accurate 2H line shape is valid only for sites with a particular CQ. It is unfeasible to use conventional CP to accurately determine 2H line shapes for all sites of a system, particularly in a protein where deuterium CQ values span ∼185 kHz range. A newly invented polarization transfer scheme, the so-called adiabatic RESPIRATION CP, (77-79) shows the capability to overcome this issue. Motivated by the better performance of this CP scheme over conventional methods, in this section, we focus on evaluating the cross-polarization uniformity that was not previously discussed in detail.
2H–13C adiabatic RESPIRATION CP was first performed on Ala crystalline powder using a pulse sequence displayed in Figure S1A. The rf field strength matching condition profiles were presented in Figure S1B. Polarization efficiency at the optimal condition is enhanced by a factor of 2.4 and 1.8 for Ala methine and methyl groups, respectively, in adiabatic RESPIRATION CP compared with conventional tangent CP. The significantly broader plateau of matching condition observed in the former case illustrates that polarization transfer is less sensitive to CP rf field strength variation (Figure S1B). As shown in a previous study, in a 2D experiment with conventional tangent 2H–13C CP, no CP condition provides accurate 2H line shapes for both of the Ala aliphatic groups concurrently. (72) In other words, the CP condition yielding an optimal 2H line shape for one Ala group fails to provide similar CP efficiency for other sites. This arises from the fact that magnetization transfer is nonuniform across the 2H powder pattern in the conventional CP approach. To evaluate the situation in adiabatic RESPIRATION CP, 2D 2H–13C correlation experiments were performed with various CP conditions. As presented in Figures S2 and S3, Ala 2H quadrupolar parameters extracted from 2D 2H–13C adiabatic RESPIRATION CP experiments remain consistent over a wide range of 2H CP rf carrier frequency offsets. Further, these values agree very well with the quadrupolar parameters obtained from the 2H one-pulse excitation experiment (Figures S2 and S4). Table S1 shows 
values of Ala 2Hα and 2Hβ extracted from 2D experiments utilizing 13C CP rf field strengths varying between 22 and 77 kHz with the matching 2H rf condition. The differences among the 
values obtained at various CP conditions are negligible, and the values agree with literature reports for the two Ala aliphatic groups. These results demonstrate that adiabatic RESPIRATION CP fulfills CP transfer uniformity over the 2H powder pattern. Further, it implies that the CP condition providing accurate 2H line shapes is insensitive to rf strengths. This performance is much improved in comparison with tangent CP, where 1 kHz rf strength variation can easily lead to 10% or more 
difference, as observed in the current work and the previous study. (72) To summarize, utilizing adiabatic RESPIRATION CP for the 2H–13C correlation step in multidimensional experiments to indirectly extract 2H line shapes provides the following advantages. First, 2H–13C CP efficiency is greatly enhanced. Second, CP transfer is uniform across the 2H powder pattern, ensuring the accuracy of the indirectly extracted 2H line shapes. Third, both CP efficiency and uniformity are much less sensitive to CP condition variation that allows for stable data collection of 3D/4D solid-state NMR spectra. These three features are essential for detecting site-specific deuterium quadrupolar information for proteins possessing deuterium CQ values typically covering the range of 0 to ∼185 kHz.
values of Ala 2Hα and 2Hβ extracted from 2D experiments utilizing 13C CP rf field strengths varying between 22 and 77 kHz with the matching 2H rf condition. The differences among the values obtained at various CP conditions are negligible, and the values agree with literature reports for the two Ala aliphatic groups. These results demonstrate that adiabatic RESPIRATION CP fulfills CP transfer uniformity over the 2H powder pattern. Further, it implies that the CP condition providing accurate 2H line shapes is insensitive to rf strengths. This performance is much improved in comparison with tangent CP, where 1 kHz rf strength variation can easily lead to 10% or more difference, as observed in the current work and the previous study. (72) To summarize, utilizing adiabatic RESPIRATION CP for the 2H–13C correlation step in multidimensional experiments to indirectly extract 2H line shapes provides the following advantages. First, 2H–13C CP efficiency is greatly enhanced. Second, CP transfer is uniform across the 2H powder pattern, ensuring the accuracy of the indirectly extracted 2H line shapes. Third, both CP efficiency and uniformity are much less sensitive to CP condition variation that allows for stable data collection of 3D/4D solid-state NMR spectra. These three features are essential for detecting site-specific deuterium quadrupolar information for proteins possessing deuterium CQ values typically covering the range of 0 to ∼185 kHz.3D 2H–13C–13C NMR Correlation Experiment Designed for Studying Protein Backbone and Side-Chain Dynamics
A 3D solid-state MAS NMR experiment utilizing adiabatic RESPIRATION CP and SPC-5 homonuclear recoupling to achieve 2H–13C and 13C–13C correlation, respectively, was designed in order to extract site-specific 2H line shapes for large systems like proteins (Figure 1A). The pulse sequence allows 2H resonances to be resolved with the assistance of 13C–13C correlation and the accurate line shapes to be extracted from the first indirect dimension. Figure 1B displays 2D planes of the 3D spectrum collected for microcrystalline GB1, where 2H line shapes are extracted for the aliphatic groups of E56. The motional averaged 2H quadrupolar parameters are readily obtained from line-shape fitting. 2H line shapes were extracted from the 3D spectrum for the majority of aliphatic groups, except those subject to resonance overlap or signal absence and aromatic rings exhibiting inefficient CP. Line-shape fitting was performed to extract motional averaged quadrupolar parameters, 
and η̅, for 140 chemical groups in GB1, and the fitting results are presented in Figure S5. The obtained 2H quadrupolar parameters convey rich information about protein dynamics that correlate with structure, local chemical environment, salt bridges, hydrogen bonds, and other interactions. To our knowledge, this is the first example that 2H motional averaged quadrupolar parameters have been determined for most sites in a protein. In the following section, discussion will first focus on the side-chain motions and then on the backbone flexibility for microcrystalline GB1.
and η̅, for 140 chemical groups in GB1, and the fitting results are presented in Figure S5. The obtained 2H quadrupolar parameters convey rich information about protein dynamics that correlate with structure, local chemical environment, salt bridges, hydrogen bonds, and other interactions. To our knowledge, this is the first example that 2H motional averaged quadrupolar parameters have been determined for most sites in a protein. In the following section, discussion will first focus on the side-chain motions and then on the backbone flexibility for microcrystalline GB1.Figure 1
Figure 1. (A) 3D 2H–13C–13C solid-state MAS NMR pulse sequence. (B) 2D 2H–13C planes of the 3D 2H–13C–13C spectrum collected for microcrystalline GB1 and the extracted 2H line shapes for the E56 residue. Experimental line shapes (black), fits (red), and fitting residuals (blue) are displayed in the right column. In the 2D planes, signals with positive and negative intensities are shown in black and green, respectively.
Lys Side-Chain Dynamics in GB1
2H line shapes for aliphatic groups of Lys were extracted from the 3D 2H–13C–13C spectrum. Table 1 shows 
and η̅ values for Lys aliphatic groups in microcrystalline GB1. The values present a large difference between 2Hα and side-chain 2H as well as among Lys residues, indicating various backbone and side-chain motions. 
and η̅ contain detailed information about the motional process, requiring analysis on a case-by-case basis depending on the physical property of a chemical group. For example, the line shape of K28 2Hβ gives a 
of 125.4 ± 1.8 kHz and a η̅ of 0.60 ± 0.05. If a reorientation in the fast-motion regime between two sites with equal population is assumed for the K28 (CH2) β group, the reorientation angle satisfies cos θ = 0.5, which is derived from the experimental η̅. Thus, a 
of 125.4 kHz corresponds to a CQ(rigid limit) equal to 200.6 kHz. This large CQ(rigid-limit) value is physically impossible for a deuteron bonded with an sp3-hybrized carbon. (64) It infers that the K28 (CH2)β group exhibits more complex dynamics than a simple two-site reorientation, resulting from the combined effect of Cα–Cβ and Cβ–Cγ librations; yet, the motional details involved in this effect are beyond the scope of the current study. Here, the focus is put on the evaluation of the 
that is proportional to order parameter derived from 
/CQ(rigid-limit). It is rational to illustrate mobility by comparing 
values of amino acid aliphatic groups in a protein, even though they do not provide motional details.
and η̅ values for Lys aliphatic groups in microcrystalline GB1. The values present a large difference between 2Hα and side-chain 2H as well as among Lys residues, indicating various backbone and side-chain motions. and η̅ contain detailed information about the motional process, requiring analysis on a case-by-case basis depending on the physical property of a chemical group. For example, the line shape of K28 2Hβ gives a of 125.4 ± 1.8 kHz and a η̅ of 0.60 ± 0.05. If a reorientation in the fast-motion regime between two sites with equal population is assumed for the K28 (CH2) β group, the reorientation angle satisfies cos θ = 0.5, which is derived from the experimental η̅. Thus, a of 125.4 kHz corresponds to a CQ(rigid limit) equal to 200.6 kHz. This large CQ(rigid-limit) value is physically impossible for a deuteron bonded with an sp3-hybrized carbon. (64) It infers that the K28 (CH2)β group exhibits more complex dynamics than a simple two-site reorientation, resulting from the combined effect of Cα–Cβ and Cβ–Cγ librations; yet, the motional details involved in this effect are beyond the scope of the current study. Here, the focus is put on the evaluation of the that is proportional to order parameter derived from /CQ(rigid-limit). It is rational to illustrate mobility by comparing values of amino acid aliphatic groups in a protein, even though they do not provide motional details.Table 1. 2H 
and η̅ Values Determined for Lys Aliphatic Sites in Microcrystalline GB1a
and η̅ Values Determined for Lys Aliphatic Sites in Microcrystalline GB1a
Table a
For deuterium undergoing large-amplitude motion, η̅ was set to zero for line-shape fitting.
Table b
The 13Cγ–13Cδ and 13Cδ−13Cξ cross peaks of K4 severely overlap with those of K10. The same 2Hδ and 2Hξ quadrupolar values were assigned to K4 and K10, which were obtained from the overlapped 2H line shapes.
Figure 2
Figure 2. (A) Crystal structure of GB1 (PDB: 2LGI) with Lys aliphatic groups shown in van der Waals spheres and coded with color scaling to 
values. Two different perspectives are shown for better visualization. (B) K50, (C) K31, (D) K13, (E) K4, (F) K10, and (G) K28 local chemical environment in GB1. The K31 (CH2)β group is color coded with cyan as the 
value is not determined. The residues having atoms within 5 Å away for the corresponding Lys residue are shown in sticks. The salt bridges between K50 and D47, E27, and K31 and the hydrogen bond between K13 side chain NH3 and G9 backbone CO are displayed by dash lines.
The GB1 crystal structure is displayed in Figure 2A with Lys residues shown in van der Waals spheres color coded by 
values. The observed large 
variation indicates that molecular dynamics differ significantly among Lys residues in protein GB1. 
values listed in Table 1 show that the K50 side chain exhibits much more restricted movement than other Lys residues. The flexibility of the amino acid backbone and side chain depends upon the local packing density, salt bridges, hydrogen bonds, and solvent accessibility and can in turn validate these phenomena. The detail of the local structure for K50 is shown in Figure 2B. This residue is in the turn connecting β3 and is exposed to the bulk solvent with more potential for interacting with water molecules (31 water molecules ≤10 Å away from its side chain based upon PDB: 2QMT (90)). It suggests that a very dynamic side chain is expected for K50 that is controversial to the experimental observation. The motion of the K50 side chain is likely restricted by a stable salt bridge formed between K50 (NH3)ζ+ and D47 Oγ with a N–O distance of 3.07 Å (PDB: 2QMT (90)). Five out of ten solid-state NMR energy minimum structures (PDB: 2LGI (91)) show that the formation of this salt bridge is allowed where the two groups are 2.73–3.09 Å apart. The existence of a stable salt bridge indicated by the rigid K50 side chain can be used to evaluate the validity of a protein structure model. Similarly, K31 and K13 exhibit moderate side-chain dynamics among the five Lys in GB1, as implied by the 
values. A salt bridge built between K31 (NH3)ζ+ and E27 Oε appears to be the reason for restricted K31 side-chain motion (Figure 2C). The O–N distance is 2.76 Å in the XRD structure (PDB: 2QMT (90)) and ≤4 Å (3.49–3.78 Å) in seven of the ten solid-state NMR minimum energy structures (PDB: 2LGI (91)), allowing the formation of a salt bridge. The 
values of K13 side-chain deuterium are very similar to those of K31. The solvent-exposure feature of K13 dictates that its side chain likely displays a large degree of mobility (Figure 2D); however, this expectation does not agree with the determined 
values. Thus, the mobility must be quenched by a salt bridge and/or a hydrogen bond. The local chemical environment suggests that no nearby negatively charged groups are available for forming a salt bridge with K13 side-chain amide group (Figure 2D). Upon the basis of the NMR (PDB: 2LGI (91)) and XRD (PDB: 2QMT (90)) structures, a hydrogen bond is likely formed between K13 (NH3)ζ+ and the G9 backbone CO groups with the O–N distance <2.5 Å (2.73 Å in XRD structure) and the O–H–N angle in the range of 122–128°. In addition, the N8 side-chain CO group serves as another potential acceptor for a hydrogen bond formed with K13 (NH3)ζ+ as indicated in the XRD structure and one of the ten NMR minimum energy structures. These hydrogen bonds overcome the desolvation energy barrier for K13 and are responsible for rigidifying its side chain. Much more dynamic side chains are observed for K4, K10, and K28. The three residues are completely extended into the solvent and have no neighboring electron acceptors available to form a stable hydrogen bond or a salt bridge (Figure 2E–G). It is noted that a salt bridge could be formed between K4 and E15 side chains based on the distance (3.17 Å) shown in the XRD structure (2QMT (90)). In addition, two intermolecular salt bridges, K4–E42 (4.81 Å) and K10–D40 (4.95 Å), exist in the X-ray crystal structure (2QMT (90)). The presence of these salt bridges seems inconsistent with the large-amplitude side-chain motions observed for K4 and K10. It is likely that the salt bridges are dynamic and are undergoing continual breaking and reformation. Alternatively, it is possible that these salt bridges are absent in the structure of the currently studied GB1 sample, which was prepared with crystallization conditions subtly different from the X-ray study.
values. The observed large variation indicates that molecular dynamics differ significantly among Lys residues in protein GB1. values listed in Table 1 show that the K50 side chain exhibits much more restricted movement than other Lys residues. The flexibility of the amino acid backbone and side chain depends upon the local packing density, salt bridges, hydrogen bonds, and solvent accessibility and can in turn validate these phenomena. The detail of the local structure for K50 is shown in Figure 2B. This residue is in the turn connecting β3 and is exposed to the bulk solvent with more potential for interacting with water molecules (31 water molecules ≤10 Å away from its side chain based upon PDB: 2QMT (90)). It suggests that a very dynamic side chain is expected for K50 that is controversial to the experimental observation. The motion of the K50 side chain is likely restricted by a stable salt bridge formed between K50 (NH3)ζ+ and D47 Oγ with a N–O distance of 3.07 Å (PDB: 2QMT (90)). Five out of ten solid-state NMR energy minimum structures (PDB: 2LGI (91)) show that the formation of this salt bridge is allowed where the two groups are 2.73–3.09 Å apart. The existence of a stable salt bridge indicated by the rigid K50 side chain can be used to evaluate the validity of a protein structure model. Similarly, K31 and K13 exhibit moderate side-chain dynamics among the five Lys in GB1, as implied by the values. A salt bridge built between K31 (NH3)ζ+ and E27 Oε appears to be the reason for restricted K31 side-chain motion (Figure 2C). The O–N distance is 2.76 Å in the XRD structure (PDB: 2QMT (90)) and ≤4 Å (3.49–3.78 Å) in seven of the ten solid-state NMR minimum energy structures (PDB: 2LGI (91)), allowing the formation of a salt bridge. The values of K13 side-chain deuterium are very similar to those of K31. The solvent-exposure feature of K13 dictates that its side chain likely displays a large degree of mobility (Figure 2D); however, this expectation does not agree with the determined values. Thus, the mobility must be quenched by a salt bridge and/or a hydrogen bond. The local chemical environment suggests that no nearby negatively charged groups are available for forming a salt bridge with K13 side-chain amide group (Figure 2D). Upon the basis of the NMR (PDB: 2LGI (91)) and XRD (PDB: 2QMT (90)) structures, a hydrogen bond is likely formed between K13 (NH3)ζ+ and the G9 backbone CO groups with the O–N distance <2.5 Å (2.73 Å in XRD structure) and the O–H–N angle in the range of 122–128°. In addition, the N8 side-chain CO group serves as another potential acceptor for a hydrogen bond formed with K13 (NH3)ζ+ as indicated in the XRD structure and one of the ten NMR minimum energy structures. These hydrogen bonds overcome the desolvation energy barrier for K13 and are responsible for rigidifying its side chain. Much more dynamic side chains are observed for K4, K10, and K28. The three residues are completely extended into the solvent and have no neighboring electron acceptors available to form a stable hydrogen bond or a salt bridge (Figure 2E–G). It is noted that a salt bridge could be formed between K4 and E15 side chains based on the distance (3.17 Å) shown in the XRD structure (2QMT (90)). In addition, two intermolecular salt bridges, K4–E42 (4.81 Å) and K10–D40 (4.95 Å), exist in the X-ray crystal structure (2QMT (90)). The presence of these salt bridges seems inconsistent with the large-amplitude side-chain motions observed for K4 and K10. It is likely that the salt bridges are dynamic and are undergoing continual breaking and reformation. Alternatively, it is possible that these salt bridges are absent in the structure of the currently studied GB1 sample, which was prepared with crystallization conditions subtly different from the X-ray study.Table 2. 2H 
and η̅ Values Determined for Asn and Asp Aliphatic Sites in Microcrystalline GB1 Proteina
and η̅ Values Determined for Asn and Asp Aliphatic Sites in Microcrystalline GB1 Proteina
Table a
For deuterium undergoing large-amplitude motion, η̅ was set to zero for line-shape fitting.
Asp and Asn Side-Chain Dynamics in GB1
Motional averaged deuterium quadrupolar coupling constants and asymmetry parameters for Asp and Asn residues are listed in Table 2. Considerable variation is observed for the backbone 2Hα and side-chain 2Hβ (Figure 3A). In general, the large 
values indicate that Asp/Asn local backbones exhibit restricted motions. The side chains are less flexible than those of Lys residues due to the shorter side chains possessing more limited spatial extension. It is shown that the side-chain dynamics are likely further quenched by hydrogen bonds, salt bridges, and desolvation for D22, D36, N37, D46, and D47. As discussed above, D47 (COO)− forms a stable salt bridge with K50 (NH3)ζ+, resulting in the stiffness of side chains for both residues (Figure 3B). Upon the basis of the chemical environment of D22 (Figure 3C), a strong hydrogen bond likely established between the D22 side chain COO– group with T25 backbone NH, where the O–N and O–H–N angles are in the range of 1.77–2.02 Å and 138–142°, respectively. Thus, the hydrogen bond contributes to the restricted side-chain motion of D22. Further, it presumes that this strong hydrogen bond formed between the loop and the α helix contributes to the structural stability of GB1. Figure 3D shows the local environment of D46. It infers that the rigid side-chain motion of D46 is due to the hydrogen bonds between COO– and the A48 backbone NH with an O–N distance of 2.66–2.71 Å and an O–H–N angle of 138–143° according to the solid-state NMR structure (PDB: 2LGI (91)). The T49 NH is another possible electron donor involved in forming a hydrogen bond, which is weaker as the O–N distance is 3.33–3.43 Å and the O–H–N angle is 144–148°. The validity of the hydrogen bonding is further supported by the absence of a competing electron acceptor for D46. It is noted that the O–N distance for the two hydrogen bonds is larger based upon the XRD structure (PDB: 2QMT (90)), 3.11 and 4.13 Å, most likely due to minor structural variation between the solid-state NMR and XRD structures originating from subtle sample preparation differences. The inflexible D36 (CH2)β group is contradictory to its solvent exposure and the lack of hydrogen bonds and a salt bridge (Figure 3E). The possible explanation is that D36 packs close to itself in the crystal lattice, deactivating the side-chain motions. This tight packing is also found to be responsible for its COO– pKa value being higher than expected. (92) The 
values of N37 imply the immobility of the side chain that resides at the edge of the open pocket formed between β strands and the α helix and is partially exposed to solvent (Figure 3F). This unique position does not explain the rigidity of the side chain. It is noted that the Y33 aromatic ring is right above N37 (NH2)δ2, allowing the formation of a N–H−π hydrogen bond between the two. Impacted by this hydrogen bond, N37 side-chain motion is significantly restricted. In fact, the N–H−π hydrogen bond is often observed in proteins and greatly contributes to structure stability. (93)
values indicate that Asp/Asn local backbones exhibit restricted motions. The side chains are less flexible than those of Lys residues due to the shorter side chains possessing more limited spatial extension. It is shown that the side-chain dynamics are likely further quenched by hydrogen bonds, salt bridges, and desolvation for D22, D36, N37, D46, and D47. As discussed above, D47 (COO)− forms a stable salt bridge with K50 (NH3)ζ+, resulting in the stiffness of side chains for both residues (Figure 3B). Upon the basis of the chemical environment of D22 (Figure 3C), a strong hydrogen bond likely established between the D22 side chain COO– group with T25 backbone NH, where the O–N and O–H–N angles are in the range of 1.77–2.02 Å and 138–142°, respectively. Thus, the hydrogen bond contributes to the restricted side-chain motion of D22. Further, it presumes that this strong hydrogen bond formed between the loop and the α helix contributes to the structural stability of GB1. Figure 3D shows the local environment of D46. It infers that the rigid side-chain motion of D46 is due to the hydrogen bonds between COO– and the A48 backbone NH with an O–N distance of 2.66–2.71 Å and an O–H–N angle of 138–143° according to the solid-state NMR structure (PDB: 2LGI (91)). The T49 NH is another possible electron donor involved in forming a hydrogen bond, which is weaker as the O–N distance is 3.33–3.43 Å and the O–H–N angle is 144–148°. The validity of the hydrogen bonding is further supported by the absence of a competing electron acceptor for D46. It is noted that the O–N distance for the two hydrogen bonds is larger based upon the XRD structure (PDB: 2QMT (90)), 3.11 and 4.13 Å, most likely due to minor structural variation between the solid-state NMR and XRD structures originating from subtle sample preparation differences. The inflexible D36 (CH2)β group is contradictory to its solvent exposure and the lack of hydrogen bonds and a salt bridge (Figure 3E). The possible explanation is that D36 packs close to itself in the crystal lattice, deactivating the side-chain motions. This tight packing is also found to be responsible for its COO– pKa value being higher than expected. (92) The values of N37 imply the immobility of the side chain that resides at the edge of the open pocket formed between β strands and the α helix and is partially exposed to solvent (Figure 3F). This unique position does not explain the rigidity of the side chain. It is noted that the Y33 aromatic ring is right above N37 (NH2)δ2, allowing the formation of a N–H−π hydrogen bond between the two. Impacted by this hydrogen bond, N37 side-chain motion is significantly restricted. In fact, the N–H−π hydrogen bond is often observed in proteins and greatly contributes to structure stability. (93)Figure 3
Figure 3. (A) Crystal structure of GB1 (PDB: 2LGI) with Asp and Asn aliphatic groups shown in van der Waals spheres and coded with color scaling to 
values. Two different perspectives are shown for better visualization. (B) D47, (C) D22, (D) D46, (E) D36, (F) N37, (G) N35, (H) D40, and (I) N8 local chemical environment in GB1. The residues having atoms within 5 Å away for the corresponding Asn or Asp residue are shown in sticks. The salt bridge between D47 and K50 and the hydrogen bonds between D22 and T25, D46 and A48/T49, and N8 and T55 are presented by dashed lines.
N8, N35, and D40 possess more flexible side chains compared with other Asn and Asp residues discussed above. Among the three, N35 is the most dynamic one as it points outward from the protein surface and interacts with surrounding solvent molecules (Figure 3G). Because of the similar situation, large-amplitude side-chain motion is expected for D40 (Figure 3H). However, it is impacted by an intermolecular interaction, where D40 forms a salt bridge with K10 (4.95 Å). (90, 92) As discussed above for K10, the salt bridge, if it exists, is highly dynamic and only slightly rigidifies the D40 and K10 side chains. The N8 side chain presents moderate flexibility due to the coexistence of opposite effects—being mobilized by the surrounding solvent and potentially restricted by hydrogen bonds (Figure 3I). Three out of the ten minimum energy solid-state NMR structures show that a hydrogen bond tends to form between N8 (NH2)δ and T55 (OH)δ1. The O–N distance and O–H–N angle are 2.56–2.59 Å and 141–148°, respectively, according to the NMR structure (PDB: 2LGI (91)). Further, K13 (NH3)ζ+ serves as another possible electron donor for hydrogen bonding with the N8 side-chain carbonyl group based upon one of the ten minimum energy solid-state NMR structures (PDB: 2LGI (91)) and the XRD structure (PDB: 2QMT (90)). The validity of these hydrogen bonds is not supported concurrently by the solid-state NMR and XRD structures, which is likely due to minor structural differences originated from the distinct protein crystallization conditions. Despite this, it is rational to conclude at this point that the hydrogen bonds discussed here likely obstruct N8 side chain motions and compensate for the solvent mobilization effect.
Glu and Gln Side-Chain Dynamics in GB1
Table 3 lists motionally averaged deuterium quadrupolar coupling constants and asymmetry parameters for Gln and Glu residues in microcrystalline GB1. The data imply that dynamics vary significantly among these residues, particularly for side chains. Due to the absence of signal, quadrupolar coupling values were not determined for several groups as indicated in Table 3. The absence of signals can happen in one of the three scenarios: inefficient 2H–13C CP transfer, significant 2H line broadening correlating with 10–5–10–6 s time scale motion, or zero net 13C–13C magnetization transfer during SPC-5 recoupling. The slow motion in the 10–5–10–6 s regime unlikely exists as both the backbone and side chains present <10–7 s motion as implied by 
values measured in this study. Further, efficient 13C–13C polarization transfer is expected for Cβ → Cα as high transfer efficiency is observed in the direction of Cα → Cβ for Q2 and E19. This rules out the third possibility that signal absence is caused by zero net 13C–13C magnetization transfer during the SPC-5 mixing period. Thus, the explanation of signal absence is that 2H–13C cross-polarization is significantly quenched by large-amplitude motion for the groups with undetermined Gln/Glu 
and η̅ values.
values measured in this study. Further, efficient 13C–13C polarization transfer is expected for Cβ → Cα as high transfer efficiency is observed in the direction of Cα → Cβ for Q2 and E19. This rules out the third possibility that signal absence is caused by zero net 13C–13C magnetization transfer during the SPC-5 mixing period. Thus, the explanation of signal absence is that 2H–13C cross-polarization is significantly quenched by large-amplitude motion for the groups with undetermined Gln/Glu and η̅ values.Table 3. 2H 
and η̅ Values Determined for Glu and Gln Aliphatic Sites in Microcrystalline GB1a
and η̅ Values Determined for Glu and Gln Aliphatic Sites in Microcrystalline GB1a
Table a
For deuterium undergoing large-amplitude motion, η̅ was set to zero for line-shape fitting.
E27 has the largest 
values among the seven residues, indicating its rigidity in GB1 microcrystals (Table 3 and Figure 4A). As discussed in the previous section, E27 COO– forms a salt bridge with K31 (NH3)ζ+ that significantly hinders side-chain motions for the two residues (Figure 4B). The moderate 
values (∼130 kHz) imply that E56 exhibits restricted side-chain motion that is uncommon for a terminal residue. Figure 4C displays the local chemical environment for E56 in GB1 crystal structure. The side chain points inward to the protein core and places COO– in a unique position that has a strong tendency to form hydrogen bonds with D40 and the K10 backbone NH. The strong–moderate hydrogen bond between E56 and K10 is expected as the O–H–N angle is close to 180° (162–176°) and the O–N distance is 2.60–3.08 Å according to the solid-state NMR structures (PDB: 2LGI (91)). In addition, a weak hydrogen bond is presumably established between E56 COO– and K10 considering the values of the O–N distance (3.00–3.49 Å) and the O–H–N angle (119–133°). Thus, E56 side-chain motion is significantly impeded by the two hydrogen bonds. It also explains why strong NMR signals are observed for E56 despite it being the C-terminal residue. Further, the two intrastrand hydrogen bonds likely have great contribution to stabilize the protein structure. The side chain of Q32 is much more dynamic than E27 and E56 illustrated by the 
values. This is consistent with the local structure, where no hydrogen bond or tight packing is indicated (Figure 4D). Although a relatively large 
(141.1 kHz) is determined for E42 (CH2)β possibly due to the backbone restriction, its (CH2)γ exhibits larger-amplitude motion in comparison with Glu/Gln (discussed above) as indicated by the undetectable 2H–13C cross-polarization. Thus, E42 (CH2)γ presents large-amplitude mobility because it is solvent exposed and actively interacts with the surrounding water molecules. The significantly motional attenuated 
(91.5 kHz) is observed for E15 2Hβ, indicating the side chain is more dynamic than those of E15, E27, Q32, and E56. This is further illustrated by that the mobility of E15 (CH2)γ is significantly enhanced leading to inefficient 2H–13C cross-polarization transfer. The dynamic E15 side chain is consistent with that it points outward from the protein surface and is unlikely to participate in the formation of a hydrogen bond (Figure 4F). However, an intramolecular salt bridge is likely formed between E15 COO– and K4 (NH3)ζ+ as supported by the XRD structure (PDB: 2QMT (90)) and E15 pH titration behavior. (92) This seems controversial to the large-amplitude side-chain motions of both E15 and K4. The most likely explanation is that the salt bridge is continually broken and reformed and, therefore, shows negligible impact on the residue side-chain motions. Q2 and E19 possess the most flexible side chains because they are exposed to solvent and have no involvement in any hydrogen bonding or salt bridging (Figure 4G and 4H).
values among the seven residues, indicating its rigidity in GB1 microcrystals (Table 3 and Figure 4A). As discussed in the previous section, E27 COO– forms a salt bridge with K31 (NH3)ζ+ that significantly hinders side-chain motions for the two residues (Figure 4B). The moderate values (∼130 kHz) imply that E56 exhibits restricted side-chain motion that is uncommon for a terminal residue. Figure 4C displays the local chemical environment for E56 in GB1 crystal structure. The side chain points inward to the protein core and places COO– in a unique position that has a strong tendency to form hydrogen bonds with D40 and the K10 backbone NH. The strong–moderate hydrogen bond between E56 and K10 is expected as the O–H–N angle is close to 180° (162–176°) and the O–N distance is 2.60–3.08 Å according to the solid-state NMR structures (PDB: 2LGI (91)). In addition, a weak hydrogen bond is presumably established between E56 COO– and K10 considering the values of the O–N distance (3.00–3.49 Å) and the O–H–N angle (119–133°). Thus, E56 side-chain motion is significantly impeded by the two hydrogen bonds. It also explains why strong NMR signals are observed for E56 despite it being the C-terminal residue. Further, the two intrastrand hydrogen bonds likely have great contribution to stabilize the protein structure. The side chain of Q32 is much more dynamic than E27 and E56 illustrated by the values. This is consistent with the local structure, where no hydrogen bond or tight packing is indicated (Figure 4D). Although a relatively large (141.1 kHz) is determined for E42 (CH2)β possibly due to the backbone restriction, its (CH2)γ exhibits larger-amplitude motion in comparison with Glu/Gln (discussed above) as indicated by the undetectable 2H–13C cross-polarization. Thus, E42 (CH2)γ presents large-amplitude mobility because it is solvent exposed and actively interacts with the surrounding water molecules. The significantly motional attenuated (91.5 kHz) is observed for E15 2Hβ, indicating the side chain is more dynamic than those of E15, E27, Q32, and E56. This is further illustrated by that the mobility of E15 (CH2)γ is significantly enhanced leading to inefficient 2H–13C cross-polarization transfer. The dynamic E15 side chain is consistent with that it points outward from the protein surface and is unlikely to participate in the formation of a hydrogen bond (Figure 4F). However, an intramolecular salt bridge is likely formed between E15 COO– and K4 (NH3)ζ+ as supported by the XRD structure (PDB: 2QMT (90)) and E15 pH titration behavior. (92) This seems controversial to the large-amplitude side-chain motions of both E15 and K4. The most likely explanation is that the salt bridge is continually broken and reformed and, therefore, shows negligible impact on the residue side-chain motions. Q2 and E19 possess the most flexible side chains because they are exposed to solvent and have no involvement in any hydrogen bonding or salt bridging (Figure 4G and 4H).Figure 4
Figure 4. (A) Crystal structure of GB1 (PDB: 2LGI) with Glu and Gln aliphatic groups shown in van der Waals spheres and coded with color scaling to 
values. Two different perspectives are shown for better visualization. (B) E27, (C) E56, (D) Q32, (E) E42, (F) E15, (G) E19, and (H) Q2 local chemical environment in GB1. Aliphatic groups having deuterium 
values undetermined are color coded with cyan. The residues having atoms within 5 Å away for the corresponding Glu or Gln residue are shown in sticks. The salt bridge between E27 and K31 and the hydrogen bond between E56 and D40/K10 are presented by dashed lines.
Thr Side-Chain Dynamics in GB1
The side-chain OH group of Thr shows a strong tendency to form a hydrogen bond, particularly with the protein backbone, as it can act as either an electron donor or acceptor. (94, 95) The hydrogen bond is expected to impede the dynamics of the Thr side chain to some extent. However, this effect seems to be substantially attenuated based on 
values determined for Thr residues in microcrystalline GB1. Table 4 lists the motional averaged deuterium quadrupolar coupling parameters for Thr aliphatic groups. It implies that the (CH2)β presents restricted motions as the 
values are close to those of backbone 2Hα for T18, T25, T44, T49, T51, and T55. The difference of 
is <8 kHz among (CH2)β groups for these residues, illustrating motions with similar amplitudes. Based on the local chemical environment, no hydrogen bond is found for side chain OH groups without ambiguity due to the divergence between reported XRD (90) and NMR (91) structures as well as between different NMR conformers. However, the existence of several hydrogen bonds is expected as supported by NMR conformers and XRD structure. A hydrogen bond likely forms between T55 (OH)δ1 and N8 (NH2)δ2, where the distance of O–N and the angle of O–H–N is 2.56–2.59 Å and 141–148°, respectively, in three of the NMR conformers (Figure 5B). The positions of T25 in five of the lowest energy NMR structures are such that its (OH)δ1 tends to form a hydrogen bond with a D22 backbone CO group (Figure 5C). The O–O distance and O–H–O angle falls in the range of 3.09–3.10 Å and 159–162°, respectively. As displayed in Figure 5D, T51 has the (OH)δ1 potentially involved in hydrogen bonding to the T49 backbone NH as shown in the XRD structure (O–O distance is 2.93 Å) and three NMR energy minimum structures (O–N distance and O–H–N angle is 3.15–3.19 Å and 124–141°, respectively). Despite the potential existence of these hydrogen bonds, the (CH2)β groups of the corresponding residues do not show further restricted motions compared to those of T18, T44, and T49.
values determined for Thr residues in microcrystalline GB1. Table 4 lists the motional averaged deuterium quadrupolar coupling parameters for Thr aliphatic groups. It implies that the (CH2)β presents restricted motions as the values are close to those of backbone 2Hα for T18, T25, T44, T49, T51, and T55. The difference of is <8 kHz among (CH2)β groups for these residues, illustrating motions with similar amplitudes. Based on the local chemical environment, no hydrogen bond is found for side chain OH groups without ambiguity due to the divergence between reported XRD (90) and NMR (91) structures as well as between different NMR conformers. However, the existence of several hydrogen bonds is expected as supported by NMR conformers and XRD structure. A hydrogen bond likely forms between T55 (OH)δ1 and N8 (NH2)δ2, where the distance of O–N and the angle of O–H–N is 2.56–2.59 Å and 141–148°, respectively, in three of the NMR conformers (Figure 5B). The positions of T25 in five of the lowest energy NMR structures are such that its (OH)δ1 tends to form a hydrogen bond with a D22 backbone CO group (Figure 5C). The O–O distance and O–H–O angle falls in the range of 3.09–3.10 Å and 159–162°, respectively. As displayed in Figure 5D, T51 has the (OH)δ1 potentially involved in hydrogen bonding to the T49 backbone NH as shown in the XRD structure (O–O distance is 2.93 Å) and three NMR energy minimum structures (O–N distance and O–H–N angle is 3.15–3.19 Å and 124–141°, respectively). Despite the potential existence of these hydrogen bonds, the (CH2)β groups of the corresponding residues do not show further restricted motions compared to those of T18, T44, and T49.The motionally reduced CQ of a Thr (CH3)γ2 originates from two processes—methyl rotation (also considered as three-site reorientation) along its C3v symmetry axis and C3v axis libration. The 
of a methyl deuterium is equal to 55.67 kHz if assuming a CQ of 167 ± 1.5 kHz (82) and an ideal tetrahedral geometry for the methyl group and is further reduced by the C3v axis libration. The (CH3)γ2
values of T18, T25, T44, T49, T51, and T55 are all close to this value but exhibit small discrepancies. These deviations could be explained by a slight departure of the methyl group geometry from the ideal tetrahedron. Such methyl geometry distortions have indeed been observed for proteins and small molecules in previous studies. (68, 96-98) For example, Ottiger and Bax determined the Hmethyl–Cmethyl–C angle deviation for methyl groups of human ubiquitin to be ±1° from 110.9°. (97) In addition, Mittermaier and Kay reported that the angle between the unique axis of the deuterium electric field gradient tensor and the C3v rotating axis (referred to as β) was 109.5°. (82) It should be noted that the Hmethyl–Cmethyl–C angle is not necessary to be identical to the β angle. If a ±1° deviation from 109.5° is assumed for the β angle, a CQ of 167 ± 1.5 kHz will yield a 
equaling 53.3–58.8 kHz for a methyl group undergoing fast rotation. The (CH3)γ2
values for T18, T25, T44, T49, and T55 determined here all fall within experimental error of this range, as shown in Table 4. One outlying group is T51 (CH3)γ2, in which the 
corresponds to its C3v axis departing at least 2.4° from that of the ideal tetrahedral geometry, under the assumption that CQ is 167 ± 1.5 kHz. (82) Nonetheless, discrepancies of this magnitude are indeed likely to occur in nature, as deviations of 1.9° for the β angle from an ideal tetrahedron have been observed for proteins by previous studies. (96, 97) The other possibilities contributing to this large discrepancy include a slight deviation of the actual CQ from 167 ± 1.5 kHz as well as any unidentified 
measurement errors. Overall, the six Thr residues exhibit very similar dynamics despite the large variation of local chemical environment; the T18 side-chain points inward to the protein core, while T49 and T44 residues are completely exposed to solvent (Figure 5E–G). It implies that Thr (CH2)β motions are dominantly restricted by the backbone and (CH3)γ2 rotations are in the fast regime and are insensitive to the local geometry as they do not require much free space.
of a methyl deuterium is equal to 55.67 kHz if assuming a CQ of 167 ± 1.5 kHz (82) and an ideal tetrahedral geometry for the methyl group and is further reduced by the C3v axis libration. The (CH3)γ2 values of T18, T25, T44, T49, T51, and T55 are all close to this value but exhibit small discrepancies. These deviations could be explained by a slight departure of the methyl group geometry from the ideal tetrahedron. Such methyl geometry distortions have indeed been observed for proteins and small molecules in previous studies. (68, 96-98) For example, Ottiger and Bax determined the Hmethyl–Cmethyl–C angle deviation for methyl groups of human ubiquitin to be ±1° from 110.9°. (97) In addition, Mittermaier and Kay reported that the angle between the unique axis of the deuterium electric field gradient tensor and the C3v rotating axis (referred to as β) was 109.5°. (82) It should be noted that the Hmethyl–Cmethyl–C angle is not necessary to be identical to the β angle. If a ±1° deviation from 109.5° is assumed for the β angle, a CQ of 167 ± 1.5 kHz will yield a equaling 53.3–58.8 kHz for a methyl group undergoing fast rotation. The (CH3)γ2 values for T18, T25, T44, T49, and T55 determined here all fall within experimental error of this range, as shown in Table 4. One outlying group is T51 (CH3)γ2, in which the corresponds to its C3v axis departing at least 2.4° from that of the ideal tetrahedral geometry, under the assumption that CQ is 167 ± 1.5 kHz. (82) Nonetheless, discrepancies of this magnitude are indeed likely to occur in nature, as deviations of 1.9° for the β angle from an ideal tetrahedron have been observed for proteins by previous studies. (96, 97) The other possibilities contributing to this large discrepancy include a slight deviation of the actual CQ from 167 ± 1.5 kHz as well as any unidentified measurement errors. Overall, the six Thr residues exhibit very similar dynamics despite the large variation of local chemical environment; the T18 side-chain points inward to the protein core, while T49 and T44 residues are completely exposed to solvent (Figure 5E–G). It implies that Thr (CH2)β motions are dominantly restricted by the backbone and (CH3)γ2 rotations are in the fast regime and are insensitive to the local geometry as they do not require much free space.The side chains of T11, T16, and T17 are much more dynamic. (CH3)γ2
values are determined to be less than 46 kHz, which are far smaller than that of a methyl group undergoing three-site reorientation as discussed above. This observation implies the presence of methyl C3v axis librations for these Thr (CH3)γ2 groups. This is typically observed for systems actively interacting with solvent (water) molecules. The local environment indicates that T11, T16, and T17 are fully extended into the solvent (Figure 5H–J). Thus, surrounding solvent significantly mobilizes the side chains of T11, T16, and T17, including the (CH2)β and (CH3)γ2 groups. It is noted that the 
is not determined for T17 (CH2)β due to weak signal as the 2H–13C cross-polarization transfer is reduced by the large-amplitude motion. Similarly, the absence of NMR signal for T53 (CH2)β and (CH3)γ2 indicates its significant mobile side chain and is consistent with solvent exposure (Figure 5K).
values are determined to be less than 46 kHz, which are far smaller than that of a methyl group undergoing three-site reorientation as discussed above. This observation implies the presence of methyl C3v axis librations for these Thr (CH3)γ2 groups. This is typically observed for systems actively interacting with solvent (water) molecules. The local environment indicates that T11, T16, and T17 are fully extended into the solvent (Figure 5H–J). Thus, surrounding solvent significantly mobilizes the side chains of T11, T16, and T17, including the (CH2)β and (CH3)γ2 groups. It is noted that the is not determined for T17 (CH2)β due to weak signal as the 2H–13C cross-polarization transfer is reduced by the large-amplitude motion. Similarly, the absence of NMR signal for T53 (CH2)β and (CH3)γ2 indicates its significant mobile side chain and is consistent with solvent exposure (Figure 5K).Table 4. 2H 
and η̅ Values Determined for Thr Aliphatic Sites in Microcrystalline GB1a
and η̅ Values Determined for Thr Aliphatic Sites in Microcrystalline GB1a
Table a
For deuterium undergoing large-amplitude motion, η̅ was set to zero for line-shape fitting.
Figure 5
Figure 5. (A) Crystal structure of GB1 (PDB: 2LGI) with Thr aliphatic groups shown in van der Waals spheres and coded with color scaling to 
values. Two different perspectives are shown for better visualization. (B) T55, (C) T25, (D) T49, (E) T18, (F) T44, (G) T49, (H) T11, (I) T16, (J) T17, and (K) T53 local chemical environment in GB1. Aliphatic groups having deuterium 
values undetermined are color coded with cyan. The residues having atoms within 5 Å away for the corresponding Thr residues are shown in sticks. The hydrogen bonds between T55 and N8, T25 and D22, and T51 and T49 are presented by dashed lines.
Side-Chain Dynamics of Nonpolar Residues in GB1
In a protein, nonpolar residues are often directed toward the molecule interior, and the side chains have no involvement in strong noncovalent interactions such as hydrogen bonding and salt bridging. The side-chain dynamics are typically dictated by local packing density and solvent accessibility. Here, we discuss the dynamics of the nonpolar residues in GB1 using the determined site-specific deuterium quadrupolar coupling parameters. Motionally averaged 
and η̅ values are shown in Table 5 for Leu and Ile residues. It implies that the (CH2)β and (CH2)γ groups in the three residues exhibit motions with similar amplitudes and are dominantly restricted by the rigid backbone. As expected, (CH2)γ groups are slightly more flexible as they extend further away from the backbone. The two (CH3)δ groups reside at the end of the side chain and are observed undergoing rotations in the fast motion regime. Again, in the current article, we do not focus the subtle differences of 
and η̅ that contain detailed dynamics information regarding motional modes. Overall, the deuterium 
values indicate that the side chain of L5 is the most rigid one among the three. This is attributed to the surrounding high packing density as the side chain is buried deeply in the protein core (Figure 6). The side chain of L12 is more dynamic compared with those of L5 and L7. The 
values of its two (CH3)δ groups are significantly reduced to 40.5 and 44.5 kHz, indicating that the C3v axes of the methyl groups undergo librations, while each deuterium is involved in fast three-site reorientation. The high mobility of the L12 side chain is a consequence of the much lower packing density and partial solvent exposure as displayed in Figure 6C. The moderately crowded local chemical environment of L7 is responsible for the side-chain dynamics with intermediate amplitude between that of L5 and L12 (Figure 6D). As discussed for Thr residues in the previous section, the 
of a methyl group is reduced to 53.3–58.8 kHz by three-site reorientation if we assume the CQ equals 167 ± 1.5 kHz (82) and the β angle possesses a 1.0° deviation from 109.5°. (82, 97) Thus, the 
of the L7 (CH3)δ2 group (59.3 ± 1.5 kHz) infers the presence of fast methyl group rotation and the immobility of the C3v axis which is equivalent to the rigidity of the Cγ–Cδ2 bond. In contrast, L5 (CH3)δ groups possess 
values larger than those for methyl groups undergoing free three-site reorientations in the rigid lattice. The (CH3)δ2
, 61.0 ± 1.6 kHz, exhibits a small discrepancy (within error), possibly due to the methyl geometry departing slightly further from the tetrahedron. The largest discrepancy is observed for the 
of L5 (CH3)δ2, which presents a β angle difference of at least 4.6° relative to an ideal tetrahedral geometry. Such a large deviation has not been reported by any previous study. It is noteworthy that this deviation could simply result from an analytical uncertainty due to the poor signal-to-noise ratio of the L5 (CH3)δ22H spectrum (Figure S5), which will require replicated data for error determination. In addition, in the three cases, different 
values are determined for the two (CH2)δ groups. It results from the restricted Cγ–Cδ bond rotation and the distinguishable local pack densities that cause appreciable different effects on the geometries and motions of the two methyl groups. I6 resides in the middle of β1, and the side chain points outward from the protein surface (Figure 6E). I6 (CH2)γ1 possesses a much smaller motional averaged 
compared with Leu (CH2)γ due to that it is exposed to solvent and less branched. It is interesting to note that the I6 methyl group is not as dynamic as L12 despite the fact that they are both exposed to solvent. This is likely due to the packing density and side-chain geometry differences correlated with the side-chain dihedral angles.
and η̅ values are shown in Table 5 for Leu and Ile residues. It implies that the (CH2)β and (CH2)γ groups in the three residues exhibit motions with similar amplitudes and are dominantly restricted by the rigid backbone. As expected, (CH2)γ groups are slightly more flexible as they extend further away from the backbone. The two (CH3)δ groups reside at the end of the side chain and are observed undergoing rotations in the fast motion regime. Again, in the current article, we do not focus the subtle differences of and η̅ that contain detailed dynamics information regarding motional modes. Overall, the deuterium values indicate that the side chain of L5 is the most rigid one among the three. This is attributed to the surrounding high packing density as the side chain is buried deeply in the protein core (Figure 6). The side chain of L12 is more dynamic compared with those of L5 and L7. The values of its two (CH3)δ groups are significantly reduced to 40.5 and 44.5 kHz, indicating that the C3v axes of the methyl groups undergo librations, while each deuterium is involved in fast three-site reorientation. The high mobility of the L12 side chain is a consequence of the much lower packing density and partial solvent exposure as displayed in Figure 6C. The moderately crowded local chemical environment of L7 is responsible for the side-chain dynamics with intermediate amplitude between that of L5 and L12 (Figure 6D). As discussed for Thr residues in the previous section, the of a methyl group is reduced to 53.3–58.8 kHz by three-site reorientation if we assume the CQ equals 167 ± 1.5 kHz (82) and the β angle possesses a 1.0° deviation from 109.5°. (82, 97) Thus, the of the L7 (CH3)δ2 group (59.3 ± 1.5 kHz) infers the presence of fast methyl group rotation and the immobility of the C3v axis which is equivalent to the rigidity of the Cγ–Cδ2 bond. In contrast, L5 (CH3)δ groups possess values larger than those for methyl groups undergoing free three-site reorientations in the rigid lattice. The (CH3)δ2, 61.0 ± 1.6 kHz, exhibits a small discrepancy (within error), possibly due to the methyl geometry departing slightly further from the tetrahedron. The largest discrepancy is observed for the of L5 (CH3)δ2, which presents a β angle difference of at least 4.6° relative to an ideal tetrahedral geometry. Such a large deviation has not been reported by any previous study. It is noteworthy that this deviation could simply result from an analytical uncertainty due to the poor signal-to-noise ratio of the L5 (CH3)δ22H spectrum (Figure S5), which will require replicated data for error determination. In addition, in the three cases, different values are determined for the two (CH2)δ groups. It results from the restricted Cγ–Cδ bond rotation and the distinguishable local pack densities that cause appreciable different effects on the geometries and motions of the two methyl groups. I6 resides in the middle of β1, and the side chain points outward from the protein surface (Figure 6E). I6 (CH2)γ1 possesses a much smaller motional averaged compared with Leu (CH2)γ due to that it is exposed to solvent and less branched. It is interesting to note that the I6 methyl group is not as dynamic as L12 despite the fact that they are both exposed to solvent. This is likely due to the packing density and side-chain geometry differences correlated with the side-chain dihedral angles.Table 5. 2H 
and η̅ Values Determined for Leu and Ile Aliphatic Sites in Microcrystalline GB1a
and η̅ Values Determined for Leu and Ile Aliphatic Sites in Microcrystalline GB1a
Table a
For deuterium undergoing large-amplitude motion, η̅ was set to zero for line-shape fitting. Quadrupolar coupling parameters were not determined for L7 (CH2)δ1 due to signal overlap.
Figure 6
Figure 6. (A) Crystal structure of GB1 (PDB: 2LGI) with Leu and Ile aliphatic groups shown in van der Waals spheres and coded with color scaling to 
values. (B) L5, (C) L12, (D) L7, and (E) I6 local chemical environment in GB1. Aliphatic groups having deuterium 
values undetermined are color coded with cyan. The residues having atoms within 5 Å away for the corresponding Leu/Ile residue are shown in sticks.
Motional averaged deuterium quadrupolar coupling parameters of Val residues in GB1 are displayed in Table 6. The parameters are not determined for the V21 side chain due to the absence of signal. It indicates that this side chain is significantly mobilized, leading to inefficient 2H → 13C cross-polarization transfer. This is consistent with the local chemical environment in the protein structure, where V21 is in a turn between β2 and the helix and fully extended to the solvent (Figure 7A and B). In contrast, V54 is observed to be the most rigid Val residue in GB1 as indicated by the 
values. The restricted motion is caused by the high packing density, as this residue points inward and is completely buried in the interior of the protein (Figure 7C). V29 exhibits dynamics with similar amplitudes as V54. The chemical environment shows that V29 resides in the middle of the well-ordered amphipathic helix (Figure 7D). Further, the geometry of the local structure and the short side-chain length likely reduce the possibility of V29 substantially interacting with solvent. V39 is in the loop between β3 and the helix and at the edge of the protein core (Figure 7E). The absence of observable 2H → 13C transfer for V39 (CH3)γ2 implies that the dynamics of this group is significantly enhanced. One possible explanation is that V39 is partially exposed to solvent as shown in Figure 7E, and only (CH3)γ2 actively interacts with solvent due to the restricted Cα–Cβ rotation. The Val dynamics discovered here further explain the polarization transfer efficiency observed for these residues in GB1 in a previous study. (84)
values. The restricted motion is caused by the high packing density, as this residue points inward and is completely buried in the interior of the protein (Figure 7C). V29 exhibits dynamics with similar amplitudes as V54. The chemical environment shows that V29 resides in the middle of the well-ordered amphipathic helix (Figure 7D). Further, the geometry of the local structure and the short side-chain length likely reduce the possibility of V29 substantially interacting with solvent. V39 is in the loop between β3 and the helix and at the edge of the protein core (Figure 7E). The absence of observable 2H → 13C transfer for V39 (CH3)γ2 implies that the dynamics of this group is significantly enhanced. One possible explanation is that V39 is partially exposed to solvent as shown in Figure 7E, and only (CH3)γ2 actively interacts with solvent due to the restricted Cα–Cβ rotation. The Val dynamics discovered here further explain the polarization transfer efficiency observed for these residues in GB1 in a previous study. (84)Table 6. 2H 
and η̅ Values Determined for Val Aliphatic Sites in Microcrystalline GB1a
and η̅ Values Determined for Val Aliphatic Sites in Microcrystalline GB1a
Table a
For deuterium undergoing large-amplitude motion, η̅ was set to zero for line-shape fitting.
Figure 7
Figure 7. (A) Crystal structure of GB1 (PDB: 2LGI) with Val aliphatic groups shown in van der Waals spheres and coded with color scaling to 
values. (B) V21, (C) V54, (D) V29, and (E) V39 local chemical environment in GB1. Aliphatic groups having deuterium 
values undetermined are color coded with cyan. The residues having atoms within 5 Å away for the corresponding Val residue are shown in sticks.
Deuterium quadrupolar coupling parameters were determined for Ala residues in GB1 as listed in Table S2. Despite the large variation in chemical environment, A20, A23, A34, and A48 possess the same 2Hβ 
value within experimental error (Figure S6). These values agree well with those of methyl groups undergoing fast three-site reorientations in rigid lattices. The small side chain methyl group makes its dynamics much less sensitive to packing density. Slightly larger 
values are observed for A26 and A24 (Table S3), likely due to similar small deviations from ideal tetrahedral geometry, the discrepancies of the CQ values, or a combination of the two (as observed for several Thr, Leu, and Ile methyl groups discussed above). It is noted that solvent exposure leads to negligible effect on the dynamics of the Ala side chain. Similarly, weak correlation between dynamics and chemical environment was previously reported for Ala methyl groups in several proteins in solution. (99)
value within experimental error (Figure S6). These values agree well with those of methyl groups undergoing fast three-site reorientations in rigid lattices. The small side chain methyl group makes its dynamics much less sensitive to packing density. Slightly larger values are observed for A26 and A24 (Table S3), likely due to similar small deviations from ideal tetrahedral geometry, the discrepancies of the CQ values, or a combination of the two (as observed for several Thr, Leu, and Ile methyl groups discussed above). It is noted that solvent exposure leads to negligible effect on the dynamics of the Ala side chain. Similarly, weak correlation between dynamics and chemical environment was previously reported for Ala methyl groups in several proteins in solution. (99)Backbone Dynamics of Microcrystalline GB1
Figure 82Hα shows the 
values for all residues in microcrystalline GB1 except for F52 of which the 
is not determined due to signal overlap. It is noted that the 2Hα 
values are close to those observed for the rigid lattice in peptides and proteins, (71, 74, 75, 100) implying that the backbone in GB1 exhibits high rigidity. Further, the detected nonzero η̅ values infer that the rigid backbone undergoes small-amplitude fluctuations and reorientations. The overall larger 2Hα 
values infer that the backbone of GB1 microcrystals exhibits motions with significantly smaller amplitudes in comparison to the side chains. Further, the small variation of the 2Hα 
indicates the high similarity of local backbone rigidity for all residues in microcrystalline GB1, despite the significant deviations of side-chain motions. This agrees with previous NMR and MD simulation studies showing that the backbone order parameter covers a narrow window. (37, 38, 83, 91, 101-103)
values for all residues in microcrystalline GB1 except for F52 of which the is not determined due to signal overlap. It is noted that the 2Hα values are close to those observed for the rigid lattice in peptides and proteins, (71, 74, 75, 100) implying that the backbone in GB1 exhibits high rigidity. Further, the detected nonzero η̅ values infer that the rigid backbone undergoes small-amplitude fluctuations and reorientations. The overall larger 2Hα values infer that the backbone of GB1 microcrystals exhibits motions with significantly smaller amplitudes in comparison to the side chains. Further, the small variation of the 2Hα indicates the high similarity of local backbone rigidity for all residues in microcrystalline GB1, despite the significant deviations of side-chain motions. This agrees with previous NMR and MD simulation studies showing that the backbone order parameter covers a narrow window. (37, 38, 83, 91, 101-103)Figure 8
Figure 8. 2Hα 
values determined for amino acid residues in microcrystalline GB1. The value for residue F52 was not determined due to signal overlap.
If a rigid-limit 2Hα CQ value of 174 kHz is assumed, (83) the backbone order parameters derived from 
/CQ(rigid-limit) cover the range of 0.82–1.0 (Figure S7), which shows a good overall agreement with 15N R1/R1ρ, CαH, and NH dipolar coupling solution-state and solid-state NMR measurements. (37, 38, 83, 91, 101-103) Particularly, the backbone order parameters obtained in the present 2H measurements agree very well with previously reported CαHα dipolar coupling data within error on the per residue basis (Figure S7). Somewhat larger discrepancies are observed between the current results and the previous relaxation and NH dipolar coupling data, which likely originates from inherent protein properties and various experimental conditions. First, the CH/CD and NH vectors are motionally distinct from each other, resulting in the discrepancies on the order parameters derived from 2Hα CQ (or CαH dipolar couplings) and NH dipolar couplings (91) as well as 2Hα and 15N solution-state NMR relaxation rates. (83) Second, discrepancies also exist between CαH vector order parameters derived from the 
values determined in this work (Figure 4S) and those determined by the previous 2H solution-state NMR relaxation studies. (83) The disagreement could be a consequence of a number of factors, including the non-negligible differences between proteins in solution and microcrystalline forms, experimental temperature differences as well as unidentified systematic uncertainties of the methodologies. Certainly, a quantitative discussion of these disagreements between studies requires complete understanding of these factors; however, this is beyond the scope of the present paper. Overall, the order parameters obtained in the current work fall within the range of 0.82–1.0 and agree well with previous studies. Interestingly, a “zigzag” pattern is observed primarily for the order parameters of the helix regions, which cannot be fully explained by the solvent exposure or packing density. It is noteworthy that the above discussion excludes the motional averaged tensor asymmetry parameters, of which the interpretation is important to characterize details of protein dynamics when using both quadrupolar and dipolar coupling measurements. (104) Slow motions at nanosecond time scales were explored for the GB1 backbone by the solid-state 15N rotating-frame relaxation rate. (37) It is known that the 2H NMR line shapes report the motional modes but not the quantitative motional rates at this dynamic regime, and 2H relaxation measurements are typically employed to assist in elucidating correlation time information. (66, 67) Backbone fluctuation/reorientation angles and rates can be extracted from 2H line shapes and relaxation times. This type of analysis requires interpretation of 
and η̅ with NMR line shape simulations on particular motional models, which is an interesting topic for future studies.
/CQ(rigid-limit) cover the range of 0.82–1.0 (Figure S7), which shows a good overall agreement with 15N R1/R1ρ, CαH, and NH dipolar coupling solution-state and solid-state NMR measurements. (37, 38, 83, 91, 101-103) Particularly, the backbone order parameters obtained in the present 2H measurements agree very well with previously reported CαHα dipolar coupling data within error on the per residue basis (Figure S7). Somewhat larger discrepancies are observed between the current results and the previous relaxation and NH dipolar coupling data, which likely originates from inherent protein properties and various experimental conditions. First, the CH/CD and NH vectors are motionally distinct from each other, resulting in the discrepancies on the order parameters derived from 2Hα CQ (or CαH dipolar couplings) and NH dipolar couplings (91) as well as 2Hα and 15N solution-state NMR relaxation rates. (83) Second, discrepancies also exist between CαH vector order parameters derived from the values determined in this work (Figure 4S) and those determined by the previous 2H solution-state NMR relaxation studies. (83) The disagreement could be a consequence of a number of factors, including the non-negligible differences between proteins in solution and microcrystalline forms, experimental temperature differences as well as unidentified systematic uncertainties of the methodologies. Certainly, a quantitative discussion of these disagreements between studies requires complete understanding of these factors; however, this is beyond the scope of the present paper. Overall, the order parameters obtained in the current work fall within the range of 0.82–1.0 and agree well with previous studies. Interestingly, a “zigzag” pattern is observed primarily for the order parameters of the helix regions, which cannot be fully explained by the solvent exposure or packing density. It is noteworthy that the above discussion excludes the motional averaged tensor asymmetry parameters, of which the interpretation is important to characterize details of protein dynamics when using both quadrupolar and dipolar coupling measurements. (104) Slow motions at nanosecond time scales were explored for the GB1 backbone by the solid-state 15N rotating-frame relaxation rate. (37) It is known that the 2H NMR line shapes report the motional modes but not the quantitative motional rates at this dynamic regime, and 2H relaxation measurements are typically employed to assist in elucidating correlation time information. (66, 67) Backbone fluctuation/reorientation angles and rates can be extracted from 2H line shapes and relaxation times. This type of analysis requires interpretation of and η̅ with NMR line shape simulations on particular motional models, which is an interesting topic for future studies.Correlations between the Dynamics of GB1 and Its Structure and Biological Function
In order to understand the side-chain dynamics of GB1 in the context of protein structure and biological function, we map out the side-chain motions across the whole protein as displayed in Figure 9. Chain lengths, degrees of branching, and polarities have distinct effects on the mobilities of different chemical groups of the side chains. Thus, the side-chain dynamics are identified as large, moderate, and small amplitude motions based on comparisons within the same type of amino acids. The analyses are conducted for amino acids presenting multiple times in GB1, including Lys, Asn/Asp, Gln/Glu, Thr, Leu, and Val, except for Ala residues that exhibit similar dynamics. On the basis of the 
values, K4, K10, N35, Q2, E15, E19, E42, T11, T16, T17, T53, L12, and V21 exhibit large-amplitude side-chain motions; K13, K28, K31, N8, D40, Q32, and V39 display moderate side-chain flexibilities; K50, D22, D36, N37, D46, D47, T18, T25, T44, T49, T51, T55, L5, L7, V29, and V54 present small-amplitude dynamics. The most dynamic side chains in GB1 are primarily observed for the β2 strand and the loop between the β1 and β2 strands (Figure 9). Previous studies revealed that these domains showed large chemical shift perturbations when binding to immunoglobulin G (IgG) and were involved in interacting with IgG. (105-109) In addition, as shown in Figure 9, side-chain motions with large to moderate amplitudes are present at the helix regions facing the β3 strand and the loop between the helix and the β3 strand, which also serve as binding domains during the interaction with IgG. The overlap between dynamic domains and binding regions suggests that the high degrees of side-chain mobilities play important roles in the contacts and interactions between GB1 and IgG. It is interesting to note that the helix regions facing the opposite direction of the mobile regions (facing way from the β3 strand) possess highly rigid side chains (Figure 9). The corresponding side chains are involved in hydrogen bonding or salt bridging and likely contribute to the stability of the protein structures, while the dynamic regions interact with IgG. The β3 strand serves as another protein binding interface, and the first residue on this β strand, E42, exhibits significant perturbed chemical shifts upon binding IgG. (106) Our results indicate that E42 is highly mobile, which likely contributes to the interaction of GB1 with IgG. It is noteworthy that the remaining residues of the β3 strand demonstrate small-amplitude side-chain motions. Among these, W43 and Y45 have the aromatic rings buried in the core of the protein, leading to the rigidity of side chains that are absent from protein interactions. The other two residues (T44 and D46) also present somewhat restricted side-chain motions. Further, the regions possessing immobile side chains include the β4 strand, the middle portion of the β1 strand, and the loop between the β3 and β4 strands, which are not involved in the interactions with IgG. It is also interesting to note that high flexibility is determined for the side chain of T53 on the β4 strand. Previous studies demonstrated that large chemical shift perturbations occurred to T53 and suggested that this residue might participate in the modulation of hydrogen bonds during protein binding. (108) Thus, the large-amplitude side-chain motions of T53 potentially assist the hydrogen bonding modulation process. In addition, the first several residues on the β1 strand possess flexible side chains as shown in Figure 9, which are expected for terminal regions. Overall, the dynamics of GB1 side chains highly correlate with its biological interactions with IgG. The residues at the binding regions exhibit large-amplitude side-chain motions, which likely facilitate protein contacts and interactions. In contrast, low side-chain mobility is primarily observed for regions that are not involved in protein binding and interactions, and the rigidity of the side chains likely assists in stabilizing the protein structure. These high correlations highlight the importance of side-chain motions in protein activities, which directly correlate to conformational entropy and have critical contributions in energetics of biological events as shown by a number of recent studies. (110-113)
values, K4, K10, N35, Q2, E15, E19, E42, T11, T16, T17, T53, L12, and V21 exhibit large-amplitude side-chain motions; K13, K28, K31, N8, D40, Q32, and V39 display moderate side-chain flexibilities; K50, D22, D36, N37, D46, D47, T18, T25, T44, T49, T51, T55, L5, L7, V29, and V54 present small-amplitude dynamics. The most dynamic side chains in GB1 are primarily observed for the β2 strand and the loop between the β1 and β2 strands (Figure 9). Previous studies revealed that these domains showed large chemical shift perturbations when binding to immunoglobulin G (IgG) and were involved in interacting with IgG. (105-109) In addition, as shown in Figure 9, side-chain motions with large to moderate amplitudes are present at the helix regions facing the β3 strand and the loop between the helix and the β3 strand, which also serve as binding domains during the interaction with IgG. The overlap between dynamic domains and binding regions suggests that the high degrees of side-chain mobilities play important roles in the contacts and interactions between GB1 and IgG. It is interesting to note that the helix regions facing the opposite direction of the mobile regions (facing way from the β3 strand) possess highly rigid side chains (Figure 9). The corresponding side chains are involved in hydrogen bonding or salt bridging and likely contribute to the stability of the protein structures, while the dynamic regions interact with IgG. The β3 strand serves as another protein binding interface, and the first residue on this β strand, E42, exhibits significant perturbed chemical shifts upon binding IgG. (106) Our results indicate that E42 is highly mobile, which likely contributes to the interaction of GB1 with IgG. It is noteworthy that the remaining residues of the β3 strand demonstrate small-amplitude side-chain motions. Among these, W43 and Y45 have the aromatic rings buried in the core of the protein, leading to the rigidity of side chains that are absent from protein interactions. The other two residues (T44 and D46) also present somewhat restricted side-chain motions. Further, the regions possessing immobile side chains include the β4 strand, the middle portion of the β1 strand, and the loop between the β3 and β4 strands, which are not involved in the interactions with IgG. It is also interesting to note that high flexibility is determined for the side chain of T53 on the β4 strand. Previous studies demonstrated that large chemical shift perturbations occurred to T53 and suggested that this residue might participate in the modulation of hydrogen bonds during protein binding. (108) Thus, the large-amplitude side-chain motions of T53 potentially assist the hydrogen bonding modulation process. In addition, the first several residues on the β1 strand possess flexible side chains as shown in Figure 9, which are expected for terminal regions. Overall, the dynamics of GB1 side chains highly correlate with its biological interactions with IgG. The residues at the binding regions exhibit large-amplitude side-chain motions, which likely facilitate protein contacts and interactions. In contrast, low side-chain mobility is primarily observed for regions that are not involved in protein binding and interactions, and the rigidity of the side chains likely assists in stabilizing the protein structure. These high correlations highlight the importance of side-chain motions in protein activities, which directly correlate to conformational entropy and have critical contributions in energetics of biological events as shown by a number of recent studies. (110-113)Figure 9
Figure 9. Protein side-chain dynamics map for microcrystalline GB1, shown in three different viewpoints. Lys, Asn, Asp, Gln, Glu, Thr, Leu, and Val residues exhibiting large, moderate, and small amplitude side-chain motions are highlighted in red, orange, and blue, respectively. The rest of the residues (gray) are not considered in this illustration.
Conclusions
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The study of atomic-level protein dynamics has lagged far behind structure determination due to the lack of high-resolution techniques. The significant role of molecular motions in protein biological function has led to increased demand for developing new approaches to probe molecular dynamics. In the current work, we present a newly designed 3D 2H–13C–13C MAS solid-state NMR pulse sequence. 2H–13C adiabatic RESPRIRATION CP implemented in the 3D pulse sequence allows for the accurate detection of 2H line shapes for deuterium-residing chemical groups in a protein in a site-specific fashion. The extracted motional averaged deuterium quadrupolar parameters provide protein backbone and side-chain dynamics for every isotopically labeled site. Here, 2H line shapes are extracted for 140 chemical groups in the protein from the 3D 2H–13C–13C MAS NMR spectrum collected for microcrystalline GB1. The obtained 
values elucidate various internal motions exhibited in the protein. The site-specific side-chain dynamics are interpreted by correlating with factors including local structure, packing density, solvent exposure, salt bridging, and hydrogen bonding. It in turn allows for validating the presence of these factors and their impact on protein dynamics and stability. Further, high correlations are demonstrated between GB1 side-chain dynamics and the biological activities. Large-amplitude side-chain motions are observed for regions that are involved in interactions with IgG. In contrast, rigid side chains are primarily found for residues that are in the core of the protein and are absent from protein binding and interactions. It infers that the high mobility of GB1 side chains likely contributes to protein contacts and binding, while the low flexibility of the side chains facilitates maintaining its structures during biological activities. These results provide critical insights into the roles of side-chain dynamics in protein biological functions. In perspective, we expect this technique to have wide applications to studies of dynamics for proteins including protein fibrils and microcrystals, as well as large membrane proteins. Further, to date, CQ has been only systematically reported for 2H at methyl and Cα sites in proteins by solution-state NMR studies. (82, 83) The current work is the first study that determines 
values for the majority of the resolved aliphatic deuterium sites in a protein. These data will enhance and extend the interpretation of 2H relaxation analysis widely used in solution-state and solid-state NMR.
values elucidate various internal motions exhibited in the protein. The site-specific side-chain dynamics are interpreted by correlating with factors including local structure, packing density, solvent exposure, salt bridging, and hydrogen bonding. It in turn allows for validating the presence of these factors and their impact on protein dynamics and stability. Further, high correlations are demonstrated between GB1 side-chain dynamics and the biological activities. Large-amplitude side-chain motions are observed for regions that are involved in interactions with IgG. In contrast, rigid side chains are primarily found for residues that are in the core of the protein and are absent from protein binding and interactions. It infers that the high mobility of GB1 side chains likely contributes to protein contacts and binding, while the low flexibility of the side chains facilitates maintaining its structures during biological activities. These results provide critical insights into the roles of side-chain dynamics in protein biological functions. In perspective, we expect this technique to have wide applications to studies of dynamics for proteins including protein fibrils and microcrystals, as well as large membrane proteins. Further, to date, CQ has been only systematically reported for 2H at methyl and Cα sites in proteins by solution-state NMR studies. (82, 83) The current work is the first study that determines values for the majority of the resolved aliphatic deuterium sites in a protein. These data will enhance and extend the interpretation of 2H relaxation analysis widely used in solution-state and solid-state NMR.Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.5b12974.
Uniform 2H magnetization transfer in 2H–13C adiabatic RESPIRATION CP (Figures S1, S2, and S3 and Table S1), Ala 2H one-pulse spectrum fit (Figure S4), GB1 2H line shape fits (Figure S5), Ala dynamics in GB1 (Table S2 and Figure S6), backbone order parameters derived from 2H measurement and CH dipolar measurement (Figure S7), GB1 2Hα η̅ values (Figure S8) (PDF)
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Acknowledgment
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This research is supported by R01-HL103999, R01-GM073770, R01-GM112845, and R21-GM107905. XS is an American Heart Association Postdoctoral Fellow (15POST25700070). We thank Dr. Deborah Berthold for help with GB1 protein sample preparation. We also thank Dr. Kristin Nuzzio, Mr. Dennis Piehl, and Ms. Lisa Della Ripa for careful reading of the manuscript.
References
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This article references 113 other publications.
- 1Teilum, K.; Olsen, J. G.; Kragelund, B. B. Cell. Mol. Life Sci. 2009, 66, 2231– 2247 DOI: 10.1007/s00018-009-0014-6Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXot12lsbc%253D&md5=caa1fa9a979d3ab2fef0e0f82b5452c0Functional aspects of protein flexibilityTeilum, Kaare; Olsen, Johan G.; Kragelund, Birthe B.Cellular and Molecular Life Sciences (2009), 66 (14), 2231-2247CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Verlag)A review. Proteins are dynamic entities, and they possess an inherent flexibility that allows them to function through mol. interactions within the cell, among cells, and even between organisms. An appreciation of the non-static nature of proteins is emerging, but to describe and incorporate this into an intuitive perception of protein function is challenging. Flexibility is of overwhelming importance for protein function, and the changes in protein structure during interactions with binding partners can be dramatic. Here, the authors address protein flexibility, focusing on protein-ligand interactions. The thermodn. involved are reviewed, and examples of structure-function studies involving exptl. detd. flexibility descriptions are presented. While much remains to be understood about protein flexibility, it is clear that it is encoded within the amino acid sequence and should be viewed as an integral part of protein structure.
- 2Henzler-Wildman, K.; Kern, D. Nature 2007, 450, 964– 972 DOI: 10.1038/nature06522Google ScholarThere is no corresponding record for this reference.
- 3Reichert, D.; Zinkevich, T.; Saalwachter, K.; Krushelnitsky, A. J. Biomol. Struct. Dyn. 2012, 30, 617– 627 DOI: 10.1080/07391102.2012.689695Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs1CjsbfN&md5=3c33cdd703eebcb170f480ed0d10f27aThe relation of the X-ray B-factor to protein dynamics: insights from recent dynamic solid-state NMR dataReichert, Detlef; Zinkevich, Tatiana; Saalwaechter, Kay; Krushelnitsky, AlexeyJournal of Biomolecular Structure and Dynamics (2012), 30 (6), 617-627CODEN: JBSDD6; ISSN:0739-1102. (Taylor & Francis Ltd.)In addressing the potential use of B-factors derived from X-ray scattering data of proteins for the understanding the (functional) dynamics of proteins, we present a comparison of B-factors of five different proteins (SH3 domain, Crh, GB1, ubiquitin and thioredoxin) with data from recent solid-state NMR expts. reflecting true (rotational) dynamics on well-defined timescales. Apart from trivial correlations involving mobile loop regions and chain termini, we find no significant correlation of B-factors with the dynamic data on any of the investigated timescales, concluding that there is no unique and general correlation of B-factors with the internal reorientational dynamics of proteins.
- 4Kuzmanic, A.; Pannu, N. S.; Zagrovic, B. Nat. Commun. 2014, 5, 3220 DOI: 10.1038/ncomms4220Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2cvivFKgsw%253D%253D&md5=99e76ed6614f1c57b46fa8917bcbcf99X-ray refinement significantly underestimates the level of microscopic heterogeneity in biomolecular crystalsKuzmanic Antonija; Zagrovic Bojan; Pannu Navraj SNature communications (2014), 5 (), 3220 ISSN:.Biomolecular X-ray structures typically provide a static, time- and ensemble-averaged view of molecular ensembles in crystals. In the absence of rigid-body motions and lattice defects, B-factors are thought to accurately reflect the structural heterogeneity of such ensembles. In order to study the effects of averaging on B-factors, we employ molecular dynamics simulations to controllably manipulate microscopic heterogeneity of a crystal containing 216 copies of villin headpiece. Using average structure factors derived from simulation, we analyse how well this heterogeneity is captured by high-resolution molecular-replacement-based model refinement. We find that both isotropic and anisotropic refined B-factors often significantly deviate from their actual values known from simulation: even at high 1.0 ÅA resolution and Rfree of 5.9%, B-factors of some well-resolved atoms underestimate their actual values even sixfold. Our results suggest that conformational averaging and inadequate treatment of correlated motion considerably influence estimation of microscopic heterogeneity via B-factors, and invite caution in their interpretation.
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- 8Krushelnitsky, A.; Reichert, D. Prog. Nucl. Magn. Reson. Spectrosc. 2005, 47, 1– 25 DOI: 10.1016/j.pnmrs.2005.04.001Google ScholarThere is no corresponding record for this reference.
- 9Watt, E. D.; Rienstra, C. M. Anal. Chem. 2014, 86, 58– 64 DOI: 10.1021/ac403956kGoogle ScholarThere is no corresponding record for this reference.
- 10Kay, L. E. J. Magn. Reson. 2005, 173, 193– 207 DOI: 10.1016/j.jmr.2004.11.021Google ScholarThere is no corresponding record for this reference.
- 11Kleckner, I. R.; Foster, M. P. Biochim. Biophys. Acta, Proteins Proteomics 2011, 1814, 942– 968 DOI: 10.1016/j.bbapap.2010.10.012Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXnsVCnsbo%253D&md5=2c047b195fd796a815e800bd162a49deAn introduction to NMR-based approaches for measuring protein dynamicsKleckner, Ian R.; Foster, Mark P.Biochimica et Biophysica Acta, Proteins and Proteomics (2011), 1814 (8), 942-968CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B. V.)A review. Proteins are inherently flexible at ambient temp. At equil., they are characterized by a set of conformations that undergo continuous exchange within a hierarchy of spatial and temporal scales ranging from nanometers to micrometers and femtoseconds to hours. Dynamic properties of proteins are essential for describing the structural bases of their biol. functions including catalysis, binding, regulation and cellular structure. NMR spectroscopy represents a powerful technique for measuring these essential features of proteins. Here the authors provide an introduction to NMR-based approaches for studying protein dynamics, highlighting eight distinct methods with recent examples, contextualized within a common exptl. and anal. framework. The selected methods are (1) Real-time NMR, (2) Exchange spectroscopy, (3) Lineshape anal., (4) CPMG relaxation dispersion, (5) Rotating frame relaxation dispersion, (6) Nuclear spin relaxation, (7) Residual dipolar coupling, (8) Paramagnetic relaxation enhancement. This article is part of a Special Issue entitled: Protein Dynamics: Exptl. and Computational Approaches.
- 12Yang, L. Q.; Sang, P.; Tao, Y.; Fu, Y. X.; Zhang, K. Q.; Xie, Y. H.; Liu, S. Q. J. Biomol. Struct. Dyn. 2014, 32, 372– 393 DOI: 10.1080/07391102.2013.770372Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXksFOms78%253D&md5=028706e72d99b9aa4779974289c72e58Protein dynamics and motions in relation to their functions: several case studies and the underlying mechanismsYang, Li-Quan; Sang, Peng; Tao, Yan; Fu, Yun-Xin; Zhang, Ke-Qin; Xie, Yue-Hui; Liu, Shu-QunJournal of Biomolecular Structure and Dynamics (2014), 32 (3), 372-393CODEN: JBSDD6; ISSN:0739-1102. (Taylor & Francis Ltd.)A review. Proteins are dynamic entities in cellular soln. with functions governed essentially by their dynamic personalities. Here, the authors review several dynamics studies on proteinase K and the HIV-1 virus envelope gp120 glycoprotein to demonstrate the importance of investigating the dynamic behaviors and mol. motions for a complete understanding of their structure-function relations. Using computer simulations and essential dynamic (ED) anal. approaches, the dynamics data obtained revealed that: (1) proteinase K has highly flexible substrate-binding site, thus supporting the induced-fit or conformational selection mechanism of substrate binding; (2) Ca2+ removal from proteinase K increases the global conformational flexibility, decreases the local flexibility of the substrate-binding region, and does not influence the thermal motion of the catalytic triad, thus explaining the exptl. detd. decreased thermostability, reduced substrate affinity, and almost unchanged catalytic activity upon Ca2+ removal; (3) substrate binding affects the large concerted motions of proteinase K, and the resulting dynamic pocket can be connected to substrate binding, orientation, and product release; (4) amino acid mutations 375 S/W and 423 I/P of HIV-1 gp120 have distinct effects on the mol. motions of gp120, facilitating the 375 S/W mutant to assume the CD4-bound conformation, while the 423 I/P mutant to prefer for the CD4-unliganded state. The mechanisms underlying protein dynamics and protein-ligand binding, including the concept of the free energy landscape (FEL) of the protein-solvent system, how the ruggedness and variability of FEL dets. protein's dynamics, and how the 3 ligand-binding models (lock-and-key, induced-fit, and conformational selection) are rationalized based on the FEL theory are discussed in depth.
- 13Karplus, M.; Kuriyan, J. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 6679– 6685 DOI: 10.1073/pnas.0408930102Google ScholarThere is no corresponding record for this reference.
- 14Lam, A. J.; St-Pierre, F.; Gong, Y.; Marshall, J. D.; Cranfill, P. J.; Baird, M. A.; McKeown, M. R.; Wiedenmann, J.; Davidson, M. W.; Schnitzer, M. J.; Tsien, R. Y.; Lin, M. Z. Nat. Methods 2012, 9, 1005– 1012 DOI: 10.1038/nmeth.2171Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhtlajs7zI&md5=4f780d655ff3cdada989b5908cefbc67Improving FRET dynamic range with bright green and red fluorescent proteinsLam, Amy J.; St-Pierre, Francois; Gong, Yiyang; Marshall, Jesse D.; Cranfill, Paula J.; Baird, Michelle A.; McKeown, Michael R.; Wiedenmann, Joerg; Davidson, Michael W.; Schnitzer, Mark J.; Tsien, Roger Y.; Lin, Michael Z.Nature Methods (2012), 9 (10), 1005-1012CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A variety of genetically encoded reporters use changes in fluorescence (or Foerster) resonance energy transfer (FRET) to report on biochem. processes in living cells. The std. genetically encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light and complex photokinetic events such as reversible photobleaching and photoconversion. We engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date and have the highest Foerster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP with these two proteins in reporters of kinase activity, small GTPase activity and transmembrane voltage significantly improves photostability, FRET dynamic range and emission ratio changes. These improvements enhance detection of transient biochem. events such as neuronal action-potential firing and RhoA activation in growth cones.
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- 22Carr, H. Y.; Purcell, E. M. Phys. Rev. 1954, 94, 630– 638 DOI: 10.1103/PhysRev.94.630Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaG2cXksFKjtA%253D%253D&md5=f729c8b5191575d29fdfd489021f05b7Effects of diffusion on free precession in nuclear magnetic resonance experimentsCarr, H. Y.; Purcell, E. M.Physical Review (1954), 94 (), 630-8CODEN: PHRVAO; ISSN:0031-899X.Nuclear resonance techniques involving free precession are examd., and a convenient variation of Hahn's spin-echo method is described (ibid. 80, 580(1950)). This variation employs a combination of pulses of different intensity or duration ("90°" and "180°" pulses). Measurements of the transverse relaxation time T2 in fluids are often severely compromised by mol. diffusion. Hahn's analysis of the effect of diffusion is reformulated and extended, and a new scheme for measuring T2 is described which, as predicted by the extended theory, largely circumvents the diffusion effect. On the other hand, the free precession technique, applied in a different way, permits a direct measurement of the mol. self-diffusion const. in suitable fluids. A measurement of the self-diffusion const. of H2O at 25° yields D = 2.5 ± 0.3 × 10-5 sq. cm./sec., in good agreement with previous detns. The effect of convection on free precession is also analyzed. A null method for measuring the longitudinal relaxation time T1, based on the unequal-pulse technique, is described.
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- 29Gobl, C.; Madl, T.; Simon, B.; Sattler, M. Prog. Nucl. Magn. Reson. Spectrosc. 2014, 80, 26– 63 DOI: 10.1016/j.pnmrs.2014.05.003Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXpslOis7g%253D&md5=b8b4e2b127b99a42e9973f960aeb7b7cNMR approaches for structural analysis of multidomain proteins and complexes in solutionGobl, Christoph; Madl, Tobias; Simon, Bernd; Sattler, MichaelProgress in Nuclear Magnetic Resonance Spectroscopy (2014), 80 (), 26-63CODEN: PNMRAT; ISSN:0079-6565. (Elsevier B.V.)A review. NMR spectroscopy is a key method for studying the structure and dynamics of (large) multidomain proteins and complexes in soln. It plays a unique role in integrated structural biol. approaches as esp. information about conformational dynamics can be readily obtained at residue resoln. Here, we review NMR techniques for such studies focusing on state-of-the-art tools and practical aspects. An efficient approach for detg. the quaternary structure of multidomain complexes starts from the structures of individual domains or subunits. The arrangement of the domains/subunits within the complex is then defined based on NMR measurements that provide information about the domain interfaces combined with (long-range) distance and orientational restraints. Aspects discussed include sample prepn., specific isotope labeling and spin labeling; detn. of binding interfaces and domain/subunit arrangements from chem. shift perturbations (CSP), nuclear Overhauser effects (NOEs), isotope editing/filtering, cross-satn., and differential line broadening; and based on paramagnetic relaxation enhancements (PRE) using covalent and sol. spin labels. Finally, the utility of complementary methods such as small-angle X-ray or neutron scattering (SAXS, SANS), ESR (EPR) or fluorescence spectroscopy techniques is discussed. The applications of NMR techniques are illustrated with studies of challenging (high mol. wt.) protein complexes.
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- 62Torchia, D. A. Annu. Rev. Biophys. Bioeng. 1984, 13, 125– 144 DOI: 10.1146/annurev.bb.13.060184.001013Google Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXkvVKnu7o%253D&md5=e8be982f7ae0fb3e1d33b924886c4751Solid state NMR studies of protein internal dynamicsTorchia, Dennis A.Annual Review of Biophysics and Bioengineering (1984), 13 (), 125-44CODEN: ABPBBK; ISSN:0084-6589.A review with 65 refs.
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- 66Jelinski, L. W. Annu. Rev. Mater. Sci. 1985, 15, 359– 377 DOI: 10.1146/annurev.ms.15.080185.002043Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXlvFKmurY%253D&md5=1b3fd9ec31842011ea6db576e7f43956Solid state deuterium NMR studies of polymer chain dynamicsJelinski, Lynn W.Annual Review of Materials Science (1985), 15 (), 359-77CODEN: ARMSCX; ISSN:0084-6600.A review with 49 refs. on solid-state D-NMR expts. used for detn. of local mol. dynamics of polymers. Also, briefly reviewed were D-NMR applications on anal. of polymeric liq. crystals, phase sepn. in polymers, and polyurethane structural anal.
- 67Hologne, M.; Hirschinger, J. Solid State Nucl. Magn. Reson. 2004, 26, 1– 10 DOI: 10.1016/S0926-2040(03)00062-6Google Scholar67https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXkt1Gjtro%253D&md5=488b9d0e6297c31e1f3664e095bc3f2fMolecular dynamics as studied by static-powder and magic-angle spinning 2H NMRHologne, Maggy; Hirschinger, JeromeSolid State Nuclear Magnetic Resonance (2004), 26 (1), 1-10CODEN: SSNRE4; ISSN:0926-2040. (Elsevier Science)The 2H NMR magic-angle spinning (MAS) technique is compared to the static-powder quadrupole echo (QE) and Jeener-Brockaert (JB) pulse sequences for a quant. investigation of mol. dynamics in solids. The linewidth of individual spinning sidebands of the one-dimensional MAS spectra are obsd. to be characteristic of the correlation time from ∼10-2 to ∼10-8 s so that the dynamic range is increased by approx. three orders of magnitude when compared to the QE expt. As a consequence, MAS 2H NMR is found to be more sensitive to the presence of an inhomogeneous distribution of correlation times than the QE and JB expts. which rely upon lineshape distortions due to anisotropic T2 and T1Q relaxation, resp. All these results are demonstrated exptl. and numerically using the two-site flip motion of di-Me sulfone and of the nitrobenzene guest in the α-p-tert-butylcalix[4]arene-nitrobenzene inclusion compd.
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- 76Wei, D.; Akbey, U. m.; Paaske, B.; Oschkinat, H.; Reif, B.; Bjerring, M.; Nielsen, N. C. J. Phys. Chem. Lett. 2011, 2, 1289– 1294 DOI: 10.1021/jz200511bGoogle Scholar76https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXlvFSqtLw%253D&md5=9804df3d0cfc0853c94e0d7e503df8b5Optimal 2H rf Pulses and 2H-13C Cross-Polarization Methods for Solid-State 2H MAS NMR of Perdeuterated ProteinsWei, Daxiu; Akbey, Umit; Paaske, Berit; Oschkinat, Hartmut; Reif, Bernd; Bjerring, Morten; Nielsen, Niels Chr.Journal of Physical Chemistry Letters (2011), 2 (11), 1289-1294CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)We present a novel concept for rf pulses and optimal control designed cross-polarization expts. for quadrupolar nuclei. The methods are demonstrated for 2H CP-MAS and 2H multiple-pulse NMR of perdeuterated proteins, for which sensitivity enhancements up to an order of magnitude are presented relative to commonly used approaches. The so-called RESPIRATION rf pulses combines the concept of short broad-band pulses with generation of pulses with large flip angles through distribution of the rf pulse over several rotor echoes. This leads to close-to-ideal rf pulses, facilitating implementation of expts. relying on the ability to realize high-performance 90 and 180° pulses, as, for example, in refocused INEPT and double-to-single quantum coherence expts., or just pulses that provide a true representation of the quadrupolar powder pattern to ext. information about the structure or dynamics. The optimal control 2H → 13C CP-MAS method demonstrates transfer efficiencies up to around 85% while being extremely robust toward rf inhomogeneity and resonance offsets.
- 77Nielsen, A. B.; Jain, S.; Ernst, M.; Meier, B. H.; Nielsen, N. C. J. Magn. Reson. 2013, 237, 147– 151 DOI: 10.1016/j.jmr.2013.09.002Google ScholarThere is no corresponding record for this reference.
- 78Jain, S.; Bjerring, M.; Nielsen, N. C. J. Phys. Chem. Lett. 2012, 3, 703– 708 DOI: 10.1021/jz3000905Google Scholar78https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XislSqtLk%253D&md5=16f412f704236445de9c532d91ebe4caEfficient and Robust Heteronuclear Cross-Polarization for High-Speed-Spinning Biological Solid-State NMR SpectroscopyJain, Sheetal; Bjerring, Morten; Nielsen, Niels Chr.Journal of Physical Chemistry Letters (2012), 3 (6), 703-708CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)The authors present a new and highly efficient approach for heteronuclear coherence transfer in solid-state NMR spectroscopy under high-speed spinning conditions. The so-called RESPIRATIONCP expt. exploits phase-alternated recoupling on only one of the two rf channels intertwined in a synchronized train of short rf pulses on both channels. The method provides significantly higher efficiencies than state-of-the art techniques including ramped and adiabatic cross-polarization expts. with long durations of intense rf irradn. At the same time, it is easier to setup exptl. and significantly more robust toward imperfections such as rf inhomogeneity, misadjustments, and sample-induced variations in the rf tuning. The method is described anal., numerically, and exptl. for biol. solids. The authors demonstrate sensitivity gains of factors of 1.3 and 1.8 for typical 1H→15N and 15N→13C transfers and a combined gain of a factor of 2-4 for a typical NCA expt. for biol. solid-state NMR.
- 79Jain, S. K.; Nielsen, A. B.; Hiller, M.; Handel, L.; Ernst, M.; Oschkinat, H.; Akbey, U.; Nielsen, N. C. Phys. Chem. Chem. Phys. 2014, 16, 2827– 2830 DOI: 10.1039/c3cp54419bGoogle ScholarThere is no corresponding record for this reference.
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- 88Delaglio, F.; Grzesiek, S.; Vuister, G. W.; Zhu, G.; Pfeifer, J.; Bax, A. J. Biomol. NMR 1995, 6, 277– 293 DOI: 10.1007/BF00197809Google Scholar88https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXhtVSmurfK&md5=a670fca5b164083e2178fafd2fb951ffNMRPipe: a multidimensional spectral processing system based on UNIX pipesDelaglio, Frank; Grzesiek, Stephan; Vuister, Geerten W.; Zhu, Guang; Pfeifer, John; Bax, AdJournal of Biomolecular NMR (1995), 6 (3), 277-93CODEN: JBNME9; ISSN:0925-2738. (ESCOM)The NMRPipe system is a UNIX software environment of processing, graphics, and anal. tools designed to meet current routine and research-oriented multidimensional processing requirements, and to anticipate and accommodate future demands and developments. The system is based on UNIX pipes, which allow programs running simultaneously to exchange streams of data under user control. In an NMRPipe processing scheme, a stream of spectral data flows through a pipeline of processing programs, each of which performs one component of the overall scheme, such as Fourier transformation or linear prediction. Complete multidimensional processing schemes are constructed as simple UNIX shell scripts. The processing modules themselves maintain and exploit accurate records of data sizes, detection modes, and calibration information in all dimensions, so that schemes can be constructed without the need to explicitly define or anticipate data sizes or storage details of real and imaginary channels during processing. The asynchronous pipeline scheme provides other substantial advantages, including high flexibility, favorable processing speeds, choice of both all-in-memory and disk-bound processing, easy adaptation to different data formats, simpler software development and maintenance, and the ability to distribute processing tasks on multi-CPU computers and computer networks.
- 89Massiot, D.; Fayon, F.; Capron, M.; King, I.; Le Calve, S.; Alonso, B.; Durand, J. O.; Bujoli, B.; Gan, Z. H.; Hoatson, G. Magn. Reson. Chem. 2002, 40, 70– 76 DOI: 10.1002/mrc.984Google Scholar89https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xlt1ajuw%253D%253D&md5=d32cb46ca90ac83a8f2d62c95a93a88fModeling one- and two-dimensional solid-state NMR spectraMassiot, Dominique; Fayon, Franck; Capron, Mickael; King, Ian; Le Calve, Stephanie; Alonso, Bruno; Durand, Jean-Olivier; Bujoli, Bruno; Gan, Zhehong; Hoatson, GinaMagnetic Resonance in Chemistry (2002), 40 (1), 70-76CODEN: MRCHEG; ISSN:0749-1581. (John Wiley & Sons Ltd.)A review. With the description of more and more complex 1- and two-dimensional NMR expts. comes the need to develop methods to make a comprehensive interpretation of the various different expts. that can be carried out on the same sample or series of related samples. The authors present some examples of modeling 1- and two-dimensional solid-state NMR spectra of I = 1/2 spin and quadrupolar nuclei, using lab.-developed software that is made available to the NMR community.
- 90Schmidt, H. L. F.; Sperling, L. J.; Gao, Y. G.; Wylie, B. J.; Boettcher, J. M.; Wilson, S. R.; Rienstra, C. A. J. Phys. Chem. B 2007, 111, 14362– 14369 DOI: 10.1021/jp075531pGoogle ScholarThere is no corresponding record for this reference.
- 91Wylie, B. J.; Sperling, L. J.; Nieuwkoop, A. J.; Franks, W. T.; Oldfield, E.; Rienstra, C. M. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 16974– 16979 DOI: 10.1073/pnas.1103728108Google ScholarThere is no corresponding record for this reference.
- 92Schmidt, H. L.; Shah, G. J.; Sperling, L. J.; Rienstra, C. M. J. Phys. Chem. Lett. 2010, 1, 1623– 1628 DOI: 10.1021/jz1004413Google Scholar92https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2srmtFehtA%253D%253D&md5=574992a689fabc298bf9be2a8fc6da69NMR Determination of Protein pK(a) Values in the Solid StateSchmidt Heather L Frericks; Shah Gautam J; Sperling Lindsay J; Rienstra Chad MThe journal of physical chemistry letters (2010), 1 (10), 1623-1628 ISSN:.Charged residues play an important role in defining key mechanistic features in many biomolecules. Determining the pK(a) values of large, membrane or fibrillar proteins can be challenging with traditional methods. In this study we show how solid-state NMR is used to monitor chemical shift changes during a pH titration for the small soluble β1 immunoglobulin binding domain of protein G. The chemical shifts of all the amino acids with charged side-chains throughout the uniformly-(13)C,(15)N-labeled protein were monitored over several samples varying in pH; pK(a) values were determined from these shifts for E27, D36, and E42, and the bounds for the pK(a) of other acidic side-chain resonances were determined. Additionally, this study shows how the calculated pK(a) values give insights into the crystal packing of the protein.
- 93Salonen, L. M.; Ellermann, M.; Diederich, F. Angew. Chem., Int. Ed. 2011, 50, 4808– 4842 DOI: 10.1002/anie.201007560Google Scholar93https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXlvVekt70%253D&md5=3684d00abc320d1ea0c71565495d5d5cAromatic rings in chemical and biological recognition: energetics and structuresSalonen, Laura M.; Ellermann, Manuel; Diederich, FrancoisAngewandte Chemie, International Edition (2011), 50 (21), 4808-4842CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. This review describes a multidimensional treatment of mol. recognition phenomena involving arom. rings in chem. and biol. systems. It summarizes new results reported since the appearance of an earlier review in 2003 in host-guest chem., biol. affinity assays and biostructural anal., data base mining in the Cambridge Structural Database (CSD) and the Protein Data Bank (PDB), and advanced computational studies. Topics addressed are arene-arene, perfluoroarene-arene, S···arom., cation-π, and anion-π interactions, as well as hydrogen bonding to π systems. The generated knowledge benefits, in particular, structure-based hit-to-lead development and lead optimization both in the pharmaceutical and in the crop protection industry. It equally facilitates the development of new advanced materials and supramol. systems, and should inspire further utilization of interactions with arom. rings to control the stereochem. outcome of synthetic transformations.
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- 100Sarkar, S. K.; Young, P. E.; Torchia, D. A. J. Am. Chem. Soc. 1986, 108, 6459– 6464 DOI: 10.1021/ja00281a002Google ScholarThere is no corresponding record for this reference.
- 101Barchi, J. J., Jr.; Grasberger, B.; Gronenborn, A. M.; Clore, G. M. Protein Sci. 1994, 3, 15– 21 DOI: 10.1002/pro.5560030103Google Scholar101https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXlt1yjtLw%253D&md5=1b2ff1da4f2eb56452cca955e9fd453bInvestigation of the backbone dynamics of the IgG-binding domain of streptococcal protein G by heteronuclear two-dimensional 1H-15N nuclear magnetic resonance spectroscopyBarchi, Joseph J., Jr.; Grasberger, Bruce; Gronenborn, Angela M.; Clore, G. MariusProtein Science (1994), 3 (1), 15-21CODEN: PRCIEI; ISSN:0961-8368.The backbone dynamics of the Ig-binding domain (B1) of streptococcal protein G, uniformly labeled with 15N, have been investigated by two-dimensional inverse detected heteronuclear 1H-15N NMR spectroscopy at 500 and 600 MHz. 15N T1, T2, and nuclear Overhauser enhancement data were obtained for all 55 backbone NH vectors of the B1 domain at both field strengths. The overall correlation time obtained from an anal. of the T1/T2 ratios was 3.3 ns at 26°. Overall, the B1 domain is a relatively rigid protein, consistent with the fact that over 95% of the residues participate in secondary structure, comprising a four-stranded sheer arranged in a -1, +3x, -1 topol., on top of which lies a single helix. Residues in the turns and loops connecting the elements of secondary structure tend to exhibit a higher degree of mobility on the picosecond time scale, as manifested by lower values of the overall order parameter. A no. of residues at the ends of the secondary structure elements display two distinct internal motions that are faster than the overall rotational correlation time: one is fast (<20 ps) and lies in the extreme narrowing limit, whereas the other is one to two orders of magnitude slower (1-3 ns) and lies outside the extreme narrowing limit. The slower motion can be explained by large-amplitude (20-40°) lumps in the N-H vectors between states with well-defined orientations that are stabilized by hydrogen bonds. In addn., residues in the helix and in the outer β-strands (particularly β-strand 2) display a small degree of chem. exchange line broadening, possibly due to a minor rotational motion of the helix relative to the sheer that curls around it.
- 102Seewald, M. J.; Pichumani, K.; Stowell, C.; Tibbals, B. V.; Regan, L.; Stone, M. J. Protein Sci. 2000, 9, 1177– 1193 DOI: 10.1110/ps.9.6.1177Google Scholar102https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXks1KqsL0%253D&md5=99d9f1cab2dfc1d2bc07a5f54bd341f6The role of backbone conformational heat capacity in protein stability: temperature dependent dynamics of the B1 domain of Streptococcal protein GSeewald, Michael J.; Pichumani, Kumar; Stowell, Cheri; Tibbals, Benjamin V.; Regan, Lynne; Stone, Martin J.Protein Science (2000), 9 (6), 1177-1193CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)The contributions of backbone NH group dynamics to the conformational heat capacity of the B1 domain of Streptococcal protein G have been estd. from the temp. dependence of 15N NMR-derived order parameters. Longitudinal (R1) and transverse (R2) relaxation rates, transverse cross-relation rates (ηxy), and steady state {1H}-15N nuclear Overhauser effects were measured at temps. of 0, 10, 20, 30, 40, and 50° for 89-100% of the backbone secondary amide nitrogen nuclei in the B1 domain. The ratio R2/ηxy was used to identify nuclei for which conformational exchange makes a significant contribution to R2. Relaxation data were fit to the extended model-free dynamics formalism, incorporating an axially sym. mol. rotational diffusion tensor. The temp. dependence of the order parameter (S2) was used to calc. the contribution of each NH group to conformational heat capacity (Cp) and a characteristic temp. (T*), representing the d. of conformational energy states accessible to each NH group. The heat capacities of the secondary structure regions of the B1 domain are significantly higher than those of comparable regions of other proteins, whereas the heat capacities of less structured regions are similar to those in other proteins. The higher local heat capacities are estd. to contribute up to ∼0.8 kJ/mol K to the total heat capacity of the B1 domain, without which the denaturation temp. would be ∼9° lower (78° rather than 87°). Thus, variation of backbone conformational heat capacity of native proteins may be a novel mechanism that contributes to high temp. stabilization of proteins.
- 103Idiyatullin, D.; Nesmelova, I.; Daragan, V. A.; Mayo, K. H. Protein Sci. 2003, 12, 914– 922 DOI: 10.1110/ps.0228703Google Scholar103https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXjtl2jt7w%253D&md5=6b5ab9f4ada5119f7396d3678d81eb53Comparison of 13CαH and 15NH backbone dynamics in protein GB1Idiyatullin, Djaudat; Nesmelova, Irina; Daragan, Vladimir A.; Mayo, Kevin H.Protein Science (2003), 12 (5), 914-922CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)This study presents a site-resolved exptl. view of backbone CαH and NH internal motions in the 56-residue Ig-binding domain of streptococcal protein G, GB1. Using 13CαH and 15NH NMR relaxation data [T1, T2, and NOE] acquired at three resonance frequencies (1H frequencies of 500, 600, and 800 MHz), spectral d. functions were calcd. as F(ω) = 2ωJ(ω) to provide a model-independent way to visualize and analyze internal motional correlation time distributions for backbone groups in GB1. Line broadening in F(ω) curves indicates the presence of nanosecond time scale internal motions (0.8 to 5 nsec) for all CαH and NH groups. Deconvolution of F(ω) curves effectively separates overall tumbling and internal motional correlation time distributions to yield more accurate order parameters than detd. by using std. model free approaches. Compared to NH groups, CαH internal motions are more broadly distributed on the nanosecond time scale, and larger CαH order parameters are related to correlated bond rotations for CαH fluctuations. Motional parameters for NH groups are more structurally correlated, with NH order parameters for example, being larger for residues in more structured regions of β-sheet and helix and generally smaller for residues in the loop and turns. This is most likely related to the observation that NH order parameters are correlated to hydrogen bonding. This study contributes to the general understanding of protein dynamics and exemplifies an alternative and easier way to analyze NMR relaxation data.
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Abstract
Figure 1
Figure 1. (A) 3D 2H–13C–13C solid-state MAS NMR pulse sequence. (B) 2D 2H–13C planes of the 3D 2H–13C–13C spectrum collected for microcrystalline GB1 and the extracted 2H line shapes for the E56 residue. Experimental line shapes (black), fits (red), and fitting residuals (blue) are displayed in the right column. In the 2D planes, signals with positive and negative intensities are shown in black and green, respectively.
Figure 2
Figure 2. (A) Crystal structure of GB1 (PDB: 2LGI) with Lys aliphatic groups shown in van der Waals spheres and coded with color scaling to
values. Two different perspectives are shown for better visualization. (B) K50, (C) K31, (D) K13, (E) K4, (F) K10, and (G) K28 local chemical environment in GB1. The K31 (CH2)β group is color coded with cyan as the value is not determined. The residues having atoms within 5 Å away for the corresponding Lys residue are shown in sticks. The salt bridges between K50 and D47, E27, and K31 and the hydrogen bond between K13 side chain NH3 and G9 backbone CO are displayed by dash lines.Figure 3
Figure 3. (A) Crystal structure of GB1 (PDB: 2LGI) with Asp and Asn aliphatic groups shown in van der Waals spheres and coded with color scaling to
values. Two different perspectives are shown for better visualization. (B) D47, (C) D22, (D) D46, (E) D36, (F) N37, (G) N35, (H) D40, and (I) N8 local chemical environment in GB1. The residues having atoms within 5 Å away for the corresponding Asn or Asp residue are shown in sticks. The salt bridge between D47 and K50 and the hydrogen bonds between D22 and T25, D46 and A48/T49, and N8 and T55 are presented by dashed lines.Figure 4
Figure 4. (A) Crystal structure of GB1 (PDB: 2LGI) with Glu and Gln aliphatic groups shown in van der Waals spheres and coded with color scaling to
values. Two different perspectives are shown for better visualization. (B) E27, (C) E56, (D) Q32, (E) E42, (F) E15, (G) E19, and (H) Q2 local chemical environment in GB1. Aliphatic groups having deuterium values undetermined are color coded with cyan. The residues having atoms within 5 Å away for the corresponding Glu or Gln residue are shown in sticks. The salt bridge between E27 and K31 and the hydrogen bond between E56 and D40/K10 are presented by dashed lines.Figure 5
Figure 5. (A) Crystal structure of GB1 (PDB: 2LGI) with Thr aliphatic groups shown in van der Waals spheres and coded with color scaling to
values. Two different perspectives are shown for better visualization. (B) T55, (C) T25, (D) T49, (E) T18, (F) T44, (G) T49, (H) T11, (I) T16, (J) T17, and (K) T53 local chemical environment in GB1. Aliphatic groups having deuterium values undetermined are color coded with cyan. The residues having atoms within 5 Å away for the corresponding Thr residues are shown in sticks. The hydrogen bonds between T55 and N8, T25 and D22, and T51 and T49 are presented by dashed lines.Figure 6
Figure 6. (A) Crystal structure of GB1 (PDB: 2LGI) with Leu and Ile aliphatic groups shown in van der Waals spheres and coded with color scaling to
values. (B) L5, (C) L12, (D) L7, and (E) I6 local chemical environment in GB1. Aliphatic groups having deuterium values undetermined are color coded with cyan. The residues having atoms within 5 Å away for the corresponding Leu/Ile residue are shown in sticks.Figure 7
Figure 7. (A) Crystal structure of GB1 (PDB: 2LGI) with Val aliphatic groups shown in van der Waals spheres and coded with color scaling to
values. (B) V21, (C) V54, (D) V29, and (E) V39 local chemical environment in GB1. Aliphatic groups having deuterium values undetermined are color coded with cyan. The residues having atoms within 5 Å away for the corresponding Val residue are shown in sticks.Figure 8
Figure 8. 2Hα
values determined for amino acid residues in microcrystalline GB1. The value for residue F52 was not determined due to signal overlap.Figure 9
Figure 9. Protein side-chain dynamics map for microcrystalline GB1, shown in three different viewpoints. Lys, Asn, Asp, Gln, Glu, Thr, Leu, and Val residues exhibiting large, moderate, and small amplitude side-chain motions are highlighted in red, orange, and blue, respectively. The rest of the residues (gray) are not considered in this illustration.
References
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- 12Yang, L. Q.; Sang, P.; Tao, Y.; Fu, Y. X.; Zhang, K. Q.; Xie, Y. H.; Liu, S. Q. J. Biomol. Struct. Dyn. 2014, 32, 372– 393 DOI: 10.1080/07391102.2013.770372Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXksFOms78%253D&md5=028706e72d99b9aa4779974289c72e58Protein dynamics and motions in relation to their functions: several case studies and the underlying mechanismsYang, Li-Quan; Sang, Peng; Tao, Yan; Fu, Yun-Xin; Zhang, Ke-Qin; Xie, Yue-Hui; Liu, Shu-QunJournal of Biomolecular Structure and Dynamics (2014), 32 (3), 372-393CODEN: JBSDD6; ISSN:0739-1102. (Taylor & Francis Ltd.)A review. Proteins are dynamic entities in cellular soln. with functions governed essentially by their dynamic personalities. Here, the authors review several dynamics studies on proteinase K and the HIV-1 virus envelope gp120 glycoprotein to demonstrate the importance of investigating the dynamic behaviors and mol. motions for a complete understanding of their structure-function relations. Using computer simulations and essential dynamic (ED) anal. approaches, the dynamics data obtained revealed that: (1) proteinase K has highly flexible substrate-binding site, thus supporting the induced-fit or conformational selection mechanism of substrate binding; (2) Ca2+ removal from proteinase K increases the global conformational flexibility, decreases the local flexibility of the substrate-binding region, and does not influence the thermal motion of the catalytic triad, thus explaining the exptl. detd. decreased thermostability, reduced substrate affinity, and almost unchanged catalytic activity upon Ca2+ removal; (3) substrate binding affects the large concerted motions of proteinase K, and the resulting dynamic pocket can be connected to substrate binding, orientation, and product release; (4) amino acid mutations 375 S/W and 423 I/P of HIV-1 gp120 have distinct effects on the mol. motions of gp120, facilitating the 375 S/W mutant to assume the CD4-bound conformation, while the 423 I/P mutant to prefer for the CD4-unliganded state. The mechanisms underlying protein dynamics and protein-ligand binding, including the concept of the free energy landscape (FEL) of the protein-solvent system, how the ruggedness and variability of FEL dets. protein's dynamics, and how the 3 ligand-binding models (lock-and-key, induced-fit, and conformational selection) are rationalized based on the FEL theory are discussed in depth.
- 13Karplus, M.; Kuriyan, J. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 6679– 6685 DOI: 10.1073/pnas.0408930102Google ScholarThere is no corresponding record for this reference.
- 14Lam, A. J.; St-Pierre, F.; Gong, Y.; Marshall, J. D.; Cranfill, P. J.; Baird, M. A.; McKeown, M. R.; Wiedenmann, J.; Davidson, M. W.; Schnitzer, M. J.; Tsien, R. Y.; Lin, M. Z. Nat. Methods 2012, 9, 1005– 1012 DOI: 10.1038/nmeth.2171Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhtlajs7zI&md5=4f780d655ff3cdada989b5908cefbc67Improving FRET dynamic range with bright green and red fluorescent proteinsLam, Amy J.; St-Pierre, Francois; Gong, Yiyang; Marshall, Jesse D.; Cranfill, Paula J.; Baird, Michelle A.; McKeown, Michael R.; Wiedenmann, Joerg; Davidson, Michael W.; Schnitzer, Mark J.; Tsien, Roger Y.; Lin, Michael Z.Nature Methods (2012), 9 (10), 1005-1012CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A variety of genetically encoded reporters use changes in fluorescence (or Foerster) resonance energy transfer (FRET) to report on biochem. processes in living cells. The std. genetically encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light and complex photokinetic events such as reversible photobleaching and photoconversion. We engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date and have the highest Foerster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP with these two proteins in reporters of kinase activity, small GTPase activity and transmembrane voltage significantly improves photostability, FRET dynamic range and emission ratio changes. These improvements enhance detection of transient biochem. events such as neuronal action-potential firing and RhoA activation in growth cones.
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- 16Nesmelov, Y. E.; Thomas, D. D. Biophys. Rev. 2010, 2, 91– 99 DOI: 10.1007/s12551-010-0032-5Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXlsVSksrw%253D&md5=324f9f0b67f707de61088b4fb823fa29Protein structural dynamics revealed by site-directed spin labeling and multifrequency EPRNesmelov, Yuri E.; Thomas, David D.Biophysical Reviews (2010), 2 (2), 91-99CODEN: BRIECG; ISSN:1867-2450. (Springer)A review. Multifrequency ESR (EPR), combined with site-directed spin labeling, is a powerful spectroscopic tool to characterize protein dynamics. The lineshape of an EPR spectrum reflects combined rotational dynamics of the spin probe's local motion within a protein, reorientations of protein domains, and overall protein tumbling. All these motions can be restricted and anisotropic, and sepn. of these motions is important for thorough characterization of protein dynamics. Multifrequency EPR distinguishes between different motions of a spin-labeled protein, due to the frequency dependence of EPR resoln. to fast and slow motion of a spin probe. This gives multifrequency EPR its unique capability to characterize protein dynamics in great detail. In this review, we analyze what makes multifrequency EPR sensitive to different rates of spin probe motion and discuss several examples of its usage to sep. spin probe dynamics and overall protein dynamics, to characterize protein backbone dynamics, and to resolve protein conformational states.
- 17Fleissner, M. R.; Bridges, M. D.; Brooks, E. K.; Cascio, D.; Kalai, T.; Hideg, K.; Hubbell, W. L. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 16241– 16246 DOI: 10.1073/pnas.1111420108Google ScholarThere is no corresponding record for this reference.
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- 22Carr, H. Y.; Purcell, E. M. Phys. Rev. 1954, 94, 630– 638 DOI: 10.1103/PhysRev.94.630Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaG2cXksFKjtA%253D%253D&md5=f729c8b5191575d29fdfd489021f05b7Effects of diffusion on free precession in nuclear magnetic resonance experimentsCarr, H. Y.; Purcell, E. M.Physical Review (1954), 94 (), 630-8CODEN: PHRVAO; ISSN:0031-899X.Nuclear resonance techniques involving free precession are examd., and a convenient variation of Hahn's spin-echo method is described (ibid. 80, 580(1950)). This variation employs a combination of pulses of different intensity or duration ("90°" and "180°" pulses). Measurements of the transverse relaxation time T2 in fluids are often severely compromised by mol. diffusion. Hahn's analysis of the effect of diffusion is reformulated and extended, and a new scheme for measuring T2 is described which, as predicted by the extended theory, largely circumvents the diffusion effect. On the other hand, the free precession technique, applied in a different way, permits a direct measurement of the mol. self-diffusion const. in suitable fluids. A measurement of the self-diffusion const. of H2O at 25° yields D = 2.5 ± 0.3 × 10-5 sq. cm./sec., in good agreement with previous detns. The effect of convection on free precession is also analyzed. A null method for measuring the longitudinal relaxation time T1, based on the unequal-pulse technique, is described.
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- 28Muhandiram, D. R.; Yamazaki, T.; Sykes, B. D.; Kay, L. E. J. Am. Chem. Soc. 1995, 117, 11536– 11544 DOI: 10.1021/ja00151a018Google ScholarThere is no corresponding record for this reference.
- 29Gobl, C.; Madl, T.; Simon, B.; Sattler, M. Prog. Nucl. Magn. Reson. Spectrosc. 2014, 80, 26– 63 DOI: 10.1016/j.pnmrs.2014.05.003Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXpslOis7g%253D&md5=b8b4e2b127b99a42e9973f960aeb7b7cNMR approaches for structural analysis of multidomain proteins and complexes in solutionGobl, Christoph; Madl, Tobias; Simon, Bernd; Sattler, MichaelProgress in Nuclear Magnetic Resonance Spectroscopy (2014), 80 (), 26-63CODEN: PNMRAT; ISSN:0079-6565. (Elsevier B.V.)A review. NMR spectroscopy is a key method for studying the structure and dynamics of (large) multidomain proteins and complexes in soln. It plays a unique role in integrated structural biol. approaches as esp. information about conformational dynamics can be readily obtained at residue resoln. Here, we review NMR techniques for such studies focusing on state-of-the-art tools and practical aspects. An efficient approach for detg. the quaternary structure of multidomain complexes starts from the structures of individual domains or subunits. The arrangement of the domains/subunits within the complex is then defined based on NMR measurements that provide information about the domain interfaces combined with (long-range) distance and orientational restraints. Aspects discussed include sample prepn., specific isotope labeling and spin labeling; detn. of binding interfaces and domain/subunit arrangements from chem. shift perturbations (CSP), nuclear Overhauser effects (NOEs), isotope editing/filtering, cross-satn., and differential line broadening; and based on paramagnetic relaxation enhancements (PRE) using covalent and sol. spin labels. Finally, the utility of complementary methods such as small-angle X-ray or neutron scattering (SAXS, SANS), ESR (EPR) or fluorescence spectroscopy techniques is discussed. The applications of NMR techniques are illustrated with studies of challenging (high mol. wt.) protein complexes.
- 30Mittermaier, A.; Kay, L. E. Science 2006, 312, 224– 228 DOI: 10.1126/science.1124964Google ScholarThere is no corresponding record for this reference.
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- 34Im, W.; Jo, S.; Kim, T. Biochim. Biophys. Acta, Biomembr. 2012, 1818, 252– 262 DOI: 10.1016/j.bbamem.2011.07.048Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XkslKrsw%253D%253D&md5=4b07dd2cf3479c393f3903d7f63871d2An ensemble dynamics approach to decipher solid-state NMR observables of membrane proteinsIm, Wonpil; Jo, Sunhwan; Kim, TaehoonBiochimica et Biophysica Acta, Biomembranes (2012), 1818 (2), 252-262CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)A review. Solid-state NMR (SSNMR) is an invaluable tool for detg. orientations of membrane proteins and peptides in lipid bilayers. Such orientational descriptions provide essential information about membrane protein functions. However, when a semi-static single conformer model is used to interpret various SSNMR observables, important dynamics information can be missing, and, sometimes, even orientational information can be misinterpreted. In addn., over the last decade, mol. dynamics (MD) simulation and semi-static SSNMR interpretation have shown certain levels of discrepancies in terms of transmembrane helix orientation and dynamics. Dynamic fitting models have recently been proposed to resolve these discrepancies by taking into account transmembrane helix whole body motions using addnl. parameters. As an alternative approach, the authors have developed SSNMR ensemble dynamics (SSNMR-ED) using multiple conformer models, which generates an ensemble of structures that satisfies the exptl. observables without any fitting parameters. In this review, various computational methods for detg. transmembrane helix orientations are discussed, and the distributions of VpuTM (from HIV-1) and WALP23 (a synthetic peptide) orientations from SSNMR-ED simulations are compared with those from MD simulations and semi-static/dynamic fitting models. Such comparisons illustrate that SSNMR-ED can be used as a general means to ext. both membrane protein structure and dynamics from the SSNMR measurements. This article is part of a Special Issue entitled: Membrane protein structure and function.
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- 37Lewandowski, J. R.; Sass, H. J.; Grzesiek, S.; Blackledge, M.; Emsley, L. J. Am. Chem. Soc. 2011, 133, 16762– 16765 DOI: 10.1021/ja206815hGoogle ScholarThere is no corresponding record for this reference.
- 38Mollica, L.; Baias, M.; Lewandowski, J. R.; Wylie, B. J.; Sperling, L. J.; Rienstra, C. M.; Emsley, L.; Blackledge, M. J. Phys. Chem. Lett. 2012, 3, 3657– 3662 DOI: 10.1021/jz3016233Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhslaksrrE&md5=4de33d158064b091ad375dd5ef2af54bAtomic-Resolution Structural Dynamics in Crystalline Proteins from NMR and Molecular SimulationMollica, Luca; Baias, Maria; Lewandowski, Jozef R.; Wylie, Benjamin J.; Sperling, Lindsay J.; Rienstra, Chad M.; Emsley, Lyndon; Blackledge, MartinJournal of Physical Chemistry Letters (2012), 3 (23), 3657-3662CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)Solid-state NMR can provide at.-resoln. information about protein motions occurring on a vast range of time scales under similar conditions to those of X-ray diffraction studies and therefore offers a highly complementary approach to characterizing the dynamic fluctuations occurring in the crystal. We compare exptl. detd. dynamic parameters, spin relaxation, chem. shifts, and dipolar couplings, to values calcd. from a 200 ns MD simulation of protein GB1 in its cryst. form, providing insight into the nature of structural dynamics occurring within the cryst. lattice. This simulation allows us to test the accuracy of commonly applied procedures for the interpretation of exptl. solid-state relaxation data in terms of dynamic modes and time scales. We discover that the potential complexity of relaxation-active motion can lead to significant under- or overestimation of dynamic amplitudes if different components are not taken into consideration.
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- 66Jelinski, L. W. Annu. Rev. Mater. Sci. 1985, 15, 359– 377 DOI: 10.1146/annurev.ms.15.080185.002043Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXlvFKmurY%253D&md5=1b3fd9ec31842011ea6db576e7f43956Solid state deuterium NMR studies of polymer chain dynamicsJelinski, Lynn W.Annual Review of Materials Science (1985), 15 (), 359-77CODEN: ARMSCX; ISSN:0084-6600.A review with 49 refs. on solid-state D-NMR expts. used for detn. of local mol. dynamics of polymers. Also, briefly reviewed were D-NMR applications on anal. of polymeric liq. crystals, phase sepn. in polymers, and polyurethane structural anal.
- 67Hologne, M.; Hirschinger, J. Solid State Nucl. Magn. Reson. 2004, 26, 1– 10 DOI: 10.1016/S0926-2040(03)00062-6Google Scholar67https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXkt1Gjtro%253D&md5=488b9d0e6297c31e1f3664e095bc3f2fMolecular dynamics as studied by static-powder and magic-angle spinning 2H NMRHologne, Maggy; Hirschinger, JeromeSolid State Nuclear Magnetic Resonance (2004), 26 (1), 1-10CODEN: SSNRE4; ISSN:0926-2040. (Elsevier Science)The 2H NMR magic-angle spinning (MAS) technique is compared to the static-powder quadrupole echo (QE) and Jeener-Brockaert (JB) pulse sequences for a quant. investigation of mol. dynamics in solids. The linewidth of individual spinning sidebands of the one-dimensional MAS spectra are obsd. to be characteristic of the correlation time from ∼10-2 to ∼10-8 s so that the dynamic range is increased by approx. three orders of magnitude when compared to the QE expt. As a consequence, MAS 2H NMR is found to be more sensitive to the presence of an inhomogeneous distribution of correlation times than the QE and JB expts. which rely upon lineshape distortions due to anisotropic T2 and T1Q relaxation, resp. All these results are demonstrated exptl. and numerically using the two-site flip motion of di-Me sulfone and of the nitrobenzene guest in the α-p-tert-butylcalix[4]arene-nitrobenzene inclusion compd.
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- 76Wei, D.; Akbey, U. m.; Paaske, B.; Oschkinat, H.; Reif, B.; Bjerring, M.; Nielsen, N. C. J. Phys. Chem. Lett. 2011, 2, 1289– 1294 DOI: 10.1021/jz200511bGoogle Scholar76https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXlvFSqtLw%253D&md5=9804df3d0cfc0853c94e0d7e503df8b5Optimal 2H rf Pulses and 2H-13C Cross-Polarization Methods for Solid-State 2H MAS NMR of Perdeuterated ProteinsWei, Daxiu; Akbey, Umit; Paaske, Berit; Oschkinat, Hartmut; Reif, Bernd; Bjerring, Morten; Nielsen, Niels Chr.Journal of Physical Chemistry Letters (2011), 2 (11), 1289-1294CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)We present a novel concept for rf pulses and optimal control designed cross-polarization expts. for quadrupolar nuclei. The methods are demonstrated for 2H CP-MAS and 2H multiple-pulse NMR of perdeuterated proteins, for which sensitivity enhancements up to an order of magnitude are presented relative to commonly used approaches. The so-called RESPIRATION rf pulses combines the concept of short broad-band pulses with generation of pulses with large flip angles through distribution of the rf pulse over several rotor echoes. This leads to close-to-ideal rf pulses, facilitating implementation of expts. relying on the ability to realize high-performance 90 and 180° pulses, as, for example, in refocused INEPT and double-to-single quantum coherence expts., or just pulses that provide a true representation of the quadrupolar powder pattern to ext. information about the structure or dynamics. The optimal control 2H → 13C CP-MAS method demonstrates transfer efficiencies up to around 85% while being extremely robust toward rf inhomogeneity and resonance offsets.
- 77Nielsen, A. B.; Jain, S.; Ernst, M.; Meier, B. H.; Nielsen, N. C. J. Magn. Reson. 2013, 237, 147– 151 DOI: 10.1016/j.jmr.2013.09.002Google ScholarThere is no corresponding record for this reference.
- 78Jain, S.; Bjerring, M.; Nielsen, N. C. J. Phys. Chem. Lett. 2012, 3, 703– 708 DOI: 10.1021/jz3000905Google Scholar78https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XislSqtLk%253D&md5=16f412f704236445de9c532d91ebe4caEfficient and Robust Heteronuclear Cross-Polarization for High-Speed-Spinning Biological Solid-State NMR SpectroscopyJain, Sheetal; Bjerring, Morten; Nielsen, Niels Chr.Journal of Physical Chemistry Letters (2012), 3 (6), 703-708CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)The authors present a new and highly efficient approach for heteronuclear coherence transfer in solid-state NMR spectroscopy under high-speed spinning conditions. The so-called RESPIRATIONCP expt. exploits phase-alternated recoupling on only one of the two rf channels intertwined in a synchronized train of short rf pulses on both channels. The method provides significantly higher efficiencies than state-of-the art techniques including ramped and adiabatic cross-polarization expts. with long durations of intense rf irradn. At the same time, it is easier to setup exptl. and significantly more robust toward imperfections such as rf inhomogeneity, misadjustments, and sample-induced variations in the rf tuning. The method is described anal., numerically, and exptl. for biol. solids. The authors demonstrate sensitivity gains of factors of 1.3 and 1.8 for typical 1H→15N and 15N→13C transfers and a combined gain of a factor of 2-4 for a typical NCA expt. for biol. solid-state NMR.
- 79Jain, S. K.; Nielsen, A. B.; Hiller, M.; Handel, L.; Ernst, M.; Oschkinat, H.; Akbey, U.; Nielsen, N. C. Phys. Chem. Chem. Phys. 2014, 16, 2827– 2830 DOI: 10.1039/c3cp54419bGoogle ScholarThere is no corresponding record for this reference.
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- 88Delaglio, F.; Grzesiek, S.; Vuister, G. W.; Zhu, G.; Pfeifer, J.; Bax, A. J. Biomol. NMR 1995, 6, 277– 293 DOI: 10.1007/BF00197809Google Scholar88https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXhtVSmurfK&md5=a670fca5b164083e2178fafd2fb951ffNMRPipe: a multidimensional spectral processing system based on UNIX pipesDelaglio, Frank; Grzesiek, Stephan; Vuister, Geerten W.; Zhu, Guang; Pfeifer, John; Bax, AdJournal of Biomolecular NMR (1995), 6 (3), 277-93CODEN: JBNME9; ISSN:0925-2738. (ESCOM)The NMRPipe system is a UNIX software environment of processing, graphics, and anal. tools designed to meet current routine and research-oriented multidimensional processing requirements, and to anticipate and accommodate future demands and developments. The system is based on UNIX pipes, which allow programs running simultaneously to exchange streams of data under user control. In an NMRPipe processing scheme, a stream of spectral data flows through a pipeline of processing programs, each of which performs one component of the overall scheme, such as Fourier transformation or linear prediction. Complete multidimensional processing schemes are constructed as simple UNIX shell scripts. The processing modules themselves maintain and exploit accurate records of data sizes, detection modes, and calibration information in all dimensions, so that schemes can be constructed without the need to explicitly define or anticipate data sizes or storage details of real and imaginary channels during processing. The asynchronous pipeline scheme provides other substantial advantages, including high flexibility, favorable processing speeds, choice of both all-in-memory and disk-bound processing, easy adaptation to different data formats, simpler software development and maintenance, and the ability to distribute processing tasks on multi-CPU computers and computer networks.
- 89Massiot, D.; Fayon, F.; Capron, M.; King, I.; Le Calve, S.; Alonso, B.; Durand, J. O.; Bujoli, B.; Gan, Z. H.; Hoatson, G. Magn. Reson. Chem. 2002, 40, 70– 76 DOI: 10.1002/mrc.984Google Scholar89https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xlt1ajuw%253D%253D&md5=d32cb46ca90ac83a8f2d62c95a93a88fModeling one- and two-dimensional solid-state NMR spectraMassiot, Dominique; Fayon, Franck; Capron, Mickael; King, Ian; Le Calve, Stephanie; Alonso, Bruno; Durand, Jean-Olivier; Bujoli, Bruno; Gan, Zhehong; Hoatson, GinaMagnetic Resonance in Chemistry (2002), 40 (1), 70-76CODEN: MRCHEG; ISSN:0749-1581. (John Wiley & Sons Ltd.)A review. With the description of more and more complex 1- and two-dimensional NMR expts. comes the need to develop methods to make a comprehensive interpretation of the various different expts. that can be carried out on the same sample or series of related samples. The authors present some examples of modeling 1- and two-dimensional solid-state NMR spectra of I = 1/2 spin and quadrupolar nuclei, using lab.-developed software that is made available to the NMR community.
- 90Schmidt, H. L. F.; Sperling, L. J.; Gao, Y. G.; Wylie, B. J.; Boettcher, J. M.; Wilson, S. R.; Rienstra, C. A. J. Phys. Chem. B 2007, 111, 14362– 14369 DOI: 10.1021/jp075531pGoogle ScholarThere is no corresponding record for this reference.
- 91Wylie, B. J.; Sperling, L. J.; Nieuwkoop, A. J.; Franks, W. T.; Oldfield, E.; Rienstra, C. M. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 16974– 16979 DOI: 10.1073/pnas.1103728108Google ScholarThere is no corresponding record for this reference.
- 92Schmidt, H. L.; Shah, G. J.; Sperling, L. J.; Rienstra, C. M. J. Phys. Chem. Lett. 2010, 1, 1623– 1628 DOI: 10.1021/jz1004413Google Scholar92https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2srmtFehtA%253D%253D&md5=574992a689fabc298bf9be2a8fc6da69NMR Determination of Protein pK(a) Values in the Solid StateSchmidt Heather L Frericks; Shah Gautam J; Sperling Lindsay J; Rienstra Chad MThe journal of physical chemistry letters (2010), 1 (10), 1623-1628 ISSN:.Charged residues play an important role in defining key mechanistic features in many biomolecules. Determining the pK(a) values of large, membrane or fibrillar proteins can be challenging with traditional methods. In this study we show how solid-state NMR is used to monitor chemical shift changes during a pH titration for the small soluble β1 immunoglobulin binding domain of protein G. The chemical shifts of all the amino acids with charged side-chains throughout the uniformly-(13)C,(15)N-labeled protein were monitored over several samples varying in pH; pK(a) values were determined from these shifts for E27, D36, and E42, and the bounds for the pK(a) of other acidic side-chain resonances were determined. Additionally, this study shows how the calculated pK(a) values give insights into the crystal packing of the protein.
- 93Salonen, L. M.; Ellermann, M.; Diederich, F. Angew. Chem., Int. Ed. 2011, 50, 4808– 4842 DOI: 10.1002/anie.201007560Google Scholar93https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXlvVekt70%253D&md5=3684d00abc320d1ea0c71565495d5d5cAromatic rings in chemical and biological recognition: energetics and structuresSalonen, Laura M.; Ellermann, Manuel; Diederich, FrancoisAngewandte Chemie, International Edition (2011), 50 (21), 4808-4842CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. This review describes a multidimensional treatment of mol. recognition phenomena involving arom. rings in chem. and biol. systems. It summarizes new results reported since the appearance of an earlier review in 2003 in host-guest chem., biol. affinity assays and biostructural anal., data base mining in the Cambridge Structural Database (CSD) and the Protein Data Bank (PDB), and advanced computational studies. Topics addressed are arene-arene, perfluoroarene-arene, S···arom., cation-π, and anion-π interactions, as well as hydrogen bonding to π systems. The generated knowledge benefits, in particular, structure-based hit-to-lead development and lead optimization both in the pharmaceutical and in the crop protection industry. It equally facilitates the development of new advanced materials and supramol. systems, and should inspire further utilization of interactions with arom. rings to control the stereochem. outcome of synthetic transformations.
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- 100Sarkar, S. K.; Young, P. E.; Torchia, D. A. J. Am. Chem. Soc. 1986, 108, 6459– 6464 DOI: 10.1021/ja00281a002Google ScholarThere is no corresponding record for this reference.
- 101Barchi, J. J., Jr.; Grasberger, B.; Gronenborn, A. M.; Clore, G. M. Protein Sci. 1994, 3, 15– 21 DOI: 10.1002/pro.5560030103Google Scholar101https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXlt1yjtLw%253D&md5=1b2ff1da4f2eb56452cca955e9fd453bInvestigation of the backbone dynamics of the IgG-binding domain of streptococcal protein G by heteronuclear two-dimensional 1H-15N nuclear magnetic resonance spectroscopyBarchi, Joseph J., Jr.; Grasberger, Bruce; Gronenborn, Angela M.; Clore, G. MariusProtein Science (1994), 3 (1), 15-21CODEN: PRCIEI; ISSN:0961-8368.The backbone dynamics of the Ig-binding domain (B1) of streptococcal protein G, uniformly labeled with 15N, have been investigated by two-dimensional inverse detected heteronuclear 1H-15N NMR spectroscopy at 500 and 600 MHz. 15N T1, T2, and nuclear Overhauser enhancement data were obtained for all 55 backbone NH vectors of the B1 domain at both field strengths. The overall correlation time obtained from an anal. of the T1/T2 ratios was 3.3 ns at 26°. Overall, the B1 domain is a relatively rigid protein, consistent with the fact that over 95% of the residues participate in secondary structure, comprising a four-stranded sheer arranged in a -1, +3x, -1 topol., on top of which lies a single helix. Residues in the turns and loops connecting the elements of secondary structure tend to exhibit a higher degree of mobility on the picosecond time scale, as manifested by lower values of the overall order parameter. A no. of residues at the ends of the secondary structure elements display two distinct internal motions that are faster than the overall rotational correlation time: one is fast (<20 ps) and lies in the extreme narrowing limit, whereas the other is one to two orders of magnitude slower (1-3 ns) and lies outside the extreme narrowing limit. The slower motion can be explained by large-amplitude (20-40°) lumps in the N-H vectors between states with well-defined orientations that are stabilized by hydrogen bonds. In addn., residues in the helix and in the outer β-strands (particularly β-strand 2) display a small degree of chem. exchange line broadening, possibly due to a minor rotational motion of the helix relative to the sheer that curls around it.
- 102Seewald, M. J.; Pichumani, K.; Stowell, C.; Tibbals, B. V.; Regan, L.; Stone, M. J. Protein Sci. 2000, 9, 1177– 1193 DOI: 10.1110/ps.9.6.1177Google Scholar102https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXks1KqsL0%253D&md5=99d9f1cab2dfc1d2bc07a5f54bd341f6The role of backbone conformational heat capacity in protein stability: temperature dependent dynamics of the B1 domain of Streptococcal protein GSeewald, Michael J.; Pichumani, Kumar; Stowell, Cheri; Tibbals, Benjamin V.; Regan, Lynne; Stone, Martin J.Protein Science (2000), 9 (6), 1177-1193CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)The contributions of backbone NH group dynamics to the conformational heat capacity of the B1 domain of Streptococcal protein G have been estd. from the temp. dependence of 15N NMR-derived order parameters. Longitudinal (R1) and transverse (R2) relaxation rates, transverse cross-relation rates (ηxy), and steady state {1H}-15N nuclear Overhauser effects were measured at temps. of 0, 10, 20, 30, 40, and 50° for 89-100% of the backbone secondary amide nitrogen nuclei in the B1 domain. The ratio R2/ηxy was used to identify nuclei for which conformational exchange makes a significant contribution to R2. Relaxation data were fit to the extended model-free dynamics formalism, incorporating an axially sym. mol. rotational diffusion tensor. The temp. dependence of the order parameter (S2) was used to calc. the contribution of each NH group to conformational heat capacity (Cp) and a characteristic temp. (T*), representing the d. of conformational energy states accessible to each NH group. The heat capacities of the secondary structure regions of the B1 domain are significantly higher than those of comparable regions of other proteins, whereas the heat capacities of less structured regions are similar to those in other proteins. The higher local heat capacities are estd. to contribute up to ∼0.8 kJ/mol K to the total heat capacity of the B1 domain, without which the denaturation temp. would be ∼9° lower (78° rather than 87°). Thus, variation of backbone conformational heat capacity of native proteins may be a novel mechanism that contributes to high temp. stabilization of proteins.
- 103Idiyatullin, D.; Nesmelova, I.; Daragan, V. A.; Mayo, K. H. Protein Sci. 2003, 12, 914– 922 DOI: 10.1110/ps.0228703Google Scholar103https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXjtl2jt7w%253D&md5=6b5ab9f4ada5119f7396d3678d81eb53Comparison of 13CαH and 15NH backbone dynamics in protein GB1Idiyatullin, Djaudat; Nesmelova, Irina; Daragan, Vladimir A.; Mayo, Kevin H.Protein Science (2003), 12 (5), 914-922CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)This study presents a site-resolved exptl. view of backbone CαH and NH internal motions in the 56-residue Ig-binding domain of streptococcal protein G, GB1. Using 13CαH and 15NH NMR relaxation data [T1, T2, and NOE] acquired at three resonance frequencies (1H frequencies of 500, 600, and 800 MHz), spectral d. functions were calcd. as F(ω) = 2ωJ(ω) to provide a model-independent way to visualize and analyze internal motional correlation time distributions for backbone groups in GB1. Line broadening in F(ω) curves indicates the presence of nanosecond time scale internal motions (0.8 to 5 nsec) for all CαH and NH groups. Deconvolution of F(ω) curves effectively separates overall tumbling and internal motional correlation time distributions to yield more accurate order parameters than detd. by using std. model free approaches. Compared to NH groups, CαH internal motions are more broadly distributed on the nanosecond time scale, and larger CαH order parameters are related to correlated bond rotations for CαH fluctuations. Motional parameters for NH groups are more structurally correlated, with NH order parameters for example, being larger for residues in more structured regions of β-sheet and helix and generally smaller for residues in the loop and turns. This is most likely related to the observation that NH order parameters are correlated to hydrogen bonding. This study contributes to the general understanding of protein dynamics and exemplifies an alternative and easier way to analyze NMR relaxation data.
- 104Schanda, P.; Huber, M.; Boisbouvier, J.; Meier, B. H.; Ernst, M. Angew. Chem., Int. Ed. 2011, 50, 11005– 11009 DOI: 10.1002/anie.201103944Google ScholarThere is no corresponding record for this reference.
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- 109Kato, K.; Lian, L. Y.; Barsukov, I. L.; Derrick, J. P.; Kim, H. H.; Tanaka, R.; Yoshino, A.; Shiraishi, M.; Shimada, I.; Arata, Y.; Roberts, G. C. K. Structure 1995, 3, 79– 85 DOI: 10.1016/S0969-2126(01)00136-8Google ScholarThere is no corresponding record for this reference.
- 110Wand, A. J. Curr. Opin. Struct. Biol. 2013, 23, 75– 81 DOI: 10.1016/j.sbi.2012.11.005Google Scholar110https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVektrzL&md5=afdc384ca87a383b523699bbc8666f38The dark energy of proteins comes to light: conformational entropy and its role in protein function revealed by NMR relaxationWand, A. JoshuaCurrent Opinion in Structural Biology (2013), 23 (1), 75-81CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. Historically it has been virtually impossible to exptl. det. the contribution of residual protein entropy to fundamental protein activities such as the binding of ligands. Recent progress has illuminated the possibility of employing NMR relaxation methods to quant. det. the role of changes in conformational entropy in mol. recognition by proteins. The method rests on using fast internal protein dynamics as a proxy. Initial results reveal a large and variable role for conformational entropy in the binding of ligands by proteins. Such a role for conformational entropy in mol. recognition has significant implications for enzymol., signal transduction, allosteric regulation and the development of protein-directed pharmaceuticals.
- 111Marlow, M. S.; Dogan, J.; Frederick, K. K.; Valentine, K. G.; Wand, A. J. Nat. Chem. Biol. 2010, 6, 352– 358 DOI: 10.1038/nchembio.347Google Scholar111https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXksFakt78%253D&md5=e9ee34fffa91f612441a77a7385015ffThe role of conformational entropy in molecular recognition by calmodulinMarlow, Michael S.; Dogan, Jakob; Frederick, Kendra K.; Valentine, Kathleen G.; Wand, A. JoshuaNature Chemical Biology (2010), 6 (5), 352-358CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)The phys. basis for high-affinity interactions involving proteins is complex and potentially involves a range of energetic contributions. Among these are changes in protein conformational entropy, which cannot yet be reliably computed from mol. structures. We have recently used changes in conformational dynamics as a proxy for changes in conformational entropy of calmodulin upon assocn. with domains from regulated proteins. The apparent change in conformational entropy was linearly related to the overall binding entropy. This view warrants a more quant. foundation. Here we calibrate an 'entropy meter' using an exptl. dynamical proxy based on NMR relaxation and show that changes in the conformational entropy of calmodulin are a significant component of the energetics of binding. Furthermore, the distribution of motion at the interface between the target domain and calmodulin is surprisingly noncomplementary. These observations promote modification of our understanding of the energetics of protein-ligand interactions.
- 112Frederick, K. K.; Marlow, M. S.; Valentine, K. G.; Wand, A. J. Nature 2007, 448, 325– 329 DOI: 10.1038/nature05959Google ScholarThere is no corresponding record for this reference.
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Supporting Information
Supporting Information
ARTICLE SECTIONSThe Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.5b12974.
Uniform 2H magnetization transfer in 2H–13C adiabatic RESPIRATION CP (Figures S1, S2, and S3 and Table S1), Ala 2H one-pulse spectrum fit (Figure S4), GB1 2H line shape fits (Figure S5), Ala dynamics in GB1 (Table S2 and Figure S6), backbone order parameters derived from 2H measurement and CH dipolar measurement (Figure S7), GB1 2Hα η̅ values (Figure S8) (PDF)
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