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Electrospray-Induced Mass Spectrometry Is Not Suitable for Determination of Peptidic Cu(II) Complexes
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Journal of the American Society for Mass Spectrometry

Cite this: J. Am. Soc. Mass Spectrom. 2021, 32, 12, 2766–2776
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https://doi.org/10.1021/jasms.1c00206
Published November 5, 2021

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Abstract

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The toolset of mass spectrometry (MS) is still expanding, and the number of metal ion complexes researched this way is growing. The Cu(II) ion forms particularly strong peptide complexes of biological interest which are frequent objects of MS studies, but quantitative aspects of some reported results are at odds with those of experiments performed in solution. Cu(II) complexes are usually characterized by fast ligand exchange rates, despite their high affinity, and we speculated that such kinetic lability could be responsible for the observed discrepancies. In order to resolve this issue, we selected peptides belonging to the ATCUN family characterized with high and thoroughly determined Cu(II) binding constants and re-estimated them using two ESI-MS techniques: standard conditions in combination with serial dilution experiments and very mild conditions for competition experiments. The sample acidification, which accompanies the electrospray formation, was simulated with the pH–jump stopped-flow technique. Our results indicate that ESI-MS should not be used for quantitative studies of Cu(II)–peptide complexes because the electrospray formation process compromises the entropic contribution to the complex stability, yielding underestimations of complex stability constants.

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Introduction

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Complexation to peptides is proposed to play important roles in Cu(II) physiology and toxicology. Cu(II) activates GHK, a wound healing factor (1,2) and α-factor, a yeast pheromone, (3,4) and is likely transported to neurons by neurokinin B. (5) It also elicits toxicity and probably gets detoxified by some variants of Aβ peptides (6,7) and protamine HP2 (8−10) and likely participates in the antifungal action of histatins, salivary antimicrobial peptides. (11) A recent study indicated that more peptides with such properties remain to be identified in human proteome. (12) Peptides have also been used extensively to model Cu(II) binding to its transport proteins, such as albumin and hCtr1 membrane transporter, (13−15) and synaptic proteins, such as prions, APP, and α-synuclein. (16,17) Two N-terminal sequence motifs, Xaa-His and Xaa-Zaa-His (where Xaa is any α-amino acid except of Cys, and Zaa is any α-amino acid except of Cys or Pro), provide the highest Cu(II) complex affinities by virtue of synergistic formation of chelate rings involving peptide nitrogen atoms (Figure 1). (18−20) The logarithmic conditional stability constants at physiological pH 7.4, log CK7.4, for Xaa-His complexes are in the range of 12.5–13, while those of Xaa-Zaa-His complexes range from 12.3 to ca. 15. (21,22) The latter are also known as ATCUN or NTS complexes. (18)

Figure 1

Figure 1. Structures of Cu(II) complexes at the N-terminal site of peptides having His in the 2nd (B) or 3rd position (A) and (C). R(n) mark side chains and the remainder of the peptide chain.

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Mass spectrometry (MS) in its many variants is one of the most versatile techniques of peptide and protein research, providing information on their composition, sequence, and post-translational modifications and via hyphenated techniques also on protein structure. It has also been widely used to determine affinity constants of molecular complexes, using a number of experimental approaches, (23−27) including studies of metal complexes. (28) A significant work was devoted to developing best conditions for quantitative determinations, including optimization of spray formation. (29,30)
Under certain conditions, MS can also be used to obtain quantitative information on metal ion complexes, e.g., Zn(II), Cu(I) or As(III). (31−34) It has also been used to determine the binding constants for Cu(II) complexes. (34−36)
One issue to consider is the rate at which the charged molecular ions are generated with respect to parallel changes in their environment. During the electrospray formation the time from injection of the sample from the capillary to the droplet fission takes a few milliseconds. (37−39) Many metal ion complexes remain intact in this time scale due to their sufficient inertness in ligand exchange reactions. (40) However, as recently demonstrated by stopped-flow and freeze-quench techniques, the formation of Cu(II) complexes with ATCUN peptides Gly-Gly-His and Aβ4–16 proceeds via a reactive intermediate step whose lifetime is in the hundred milliseconds range. (20,41) The Cu(II) ion in this species is partially coordinated to the amine and imidazole nitrogens only (Figure 1A) and is considered to be kinetically labile. Such lability may in turn affect the apparent binding constant determination by exposing partially folded/coordinated complexes to the gas phase. Moreover, alternative complex stoichiometries can occur. For example, Matsumoto et al. discovered Cu(II) complexes that emerged rapidly in the gas phase but were not present in solution. (42)
In addition, the presence of stable, e.g., “physiological”, conditions in the gas phase is very debatable. Protein and peptide structures are maintained through covalent bonds, hydrogen bonds, and hydrophobic interactions with a strong contribution from the solvent entropy. (43,44) The pH and ionic strength cannot be defined anymore for the molecule after its transition to gas phase. Protonation states are altered (45) and hydrophobic interactions get lost altogether, along with the evaporation of solvating water molecules. (46) In contrast, electrostatic interactions, hydrogen bonds and van der Waals interactions are enhanced in the gas phase, prompting the loss of native structure. (46) The MS measurement time scale is generally considered as too short for the protein or peptide unfolding, because it requires a concerted breaking of multiple noncovalent bonds. (47,48) Some proteins may actually unfold in less than 1 ms, however. (49) Metal binding to a peptide contributes only a few coordination bonds, typically two to six, and perhaps several weaker interactions in the second coordination sphere. The loss of even one of them due to gas phase conditions will have a profound effect on the complex stability. This is particularly important for peptidic Cu(II) complexes in which the entropic contribution to stability is very significant. (50,51)
Next, the equilibrium could be shifted because the pH of electrospray droplets might be different from that set in the sample prior to the analysis. Finding an appropriate buffer for ESI-MS studies is a difficult task. (52) Most buffers that maintain physiological pH do not qualify because their ionic character suppresses the signal. Ammonium acetate is commonly used as a “buffer” for so-called native ESI-MS experiments due to volatility of neutral forms of its components, acetic acid and ammonia, which form upon the spray evaporation and do not contribute to ionic noise. However, as pointed out by Konermann, ammonium acetate is not really a buffer at pH 7. (52) It has two buffering areas around the pK values of its components, at 4.75 ± 1 for acetate and 9.25 ± 1 for ammonia. Hence, the rising number of H+ ions produced by electric field in shrinking solvent droplets in the positive ion measurement mode rapidly decreases the droplet pH, down to the acetate buffering range of 4.75 ± 1. Under these circumstances there is no straightforward way of determining how exactly the pH has changed during the measurement. This poses a serious problem for quantitative analysis because the binding constants may change drastically with pH (see Table 1). The situation is further aggravated when mixtures of water and organic solvents are used, making the pH even more difficult to ascertain. (53) The discrepant effects of droplet acidification and other effects mentioned above on the characterization of metal ion complexes have been noted, e.g., for relatively weak complexes of alkaline earth metal ions with EDTA, (54) or lanthanide complexes with acetate, (55) but also for very tightly bound species, e.g., the Bi3+ complex of transferrin. (56)
Table 1. Comparison of Conditional Cu(II) Binding Constants of the Studied Peptides at pH = 7.4 (CK7.4), Compared to These Constants Determined Previously by Other Methods and Publisheda
peptide sequence (name)published log CK7.4blog Kd ± SD from serial dilution ESI-MS experiment in this studylog ratio log Kd (ESI-MS) – log CK7.4 (published)range of concentrations in this study (μM)
DTHFPI-NH2(hepc6)14.7c7.6 ± 0.7–7.1100–0.01
MNH-NH214.5d6.3 ± 0.4–8.2100–2
FRHDSG (Aβ4–9)14.2e6.9 ± 0.9–5.3100–1
FRHDSGYEVHHQK-NH2 (Aβ4–16)13.5f7.0 ± 0.2–6.5100–0.5
MDH-NH213.1d7.1 ± 0.6–6.0100–1
GGH12.2g6.0 ± 0.5–6.2100–10
a

Standard deviation of ESI-MS experimental values are given in parentheses. Logarithmic values are given for better clarity. The published log CK7.4 values were determined by potentiometry and corroborated by spectroscopic methods.

b

The SD values were considerably less than 0.1 log unit in all cases.

c

Reference (63).

d

Reference (64).

e

Reference (65).

f

Reference (6).

g

Reference (21).

Other available buffers offer little alternative. Ammonium bicarbonate has pKa = 6.4 but is unstable and prone to CO2 evolution, while common biologically friendly buffers, like Tris or HEPES, contain nonvolatile cations and anions, resulting in a poor MS data quality.
Furthermore, redox-prone metal complexes, including the Cu(II) species, may undergo reduction in the gas phase. This effect is independent of the method of ionization, as such behavior was reported in both electrospray and plasma desorption studies. (57,58) It is difficult to monitor and can influence quantitative studies, as the copper oxidation state is crucial for the complex formation and stability. For example, the ESI-MS experiments on the Cu(II)–GHK peptide complex yielded a mixture of Cu(II) and Cu(I) species in a proportion depending on the electrostatic potential in the ion source. (59) Tsybizova et al. listed three possible mechanisms for Cu(II) reduction: reduction on the capillary walls, desolvation with electron transfer and reduction while still in the solution. (60) Reduction was more likely to occur when the Cu(II) ion was coordinated by no more than two atoms.
The unpredictability of issues with reduction and pH shift is aggravated by an uncertainty of the measurement time scale which depends on the droplet size. As a general rule, the bigger the droplet is, the longer time it takes to dissipate, extending the opportunity for unwanted processes to occur. (61)
In our laboratory practice, we frequently recorded ESI-MS spectra of Cu(II) complexes of peptides. Nearly always strong signals of unbound peptide were observed, although they were not present in equilibrium (see Figure 2 for examples). This strongly indicated that complex dissociation occurred in the mass spectrometer, yielding artifacts. On the other hand, ESI-MS is often treated, e.g. by less experienced researchers as if it faithfully reflected the solution equilibria. The goal of our study was therefore to systematically explore the issue of a systematic error in attempts to study Cu(II)/peptide complexes quantitatively by ESI-MS. In order to achieve it, we performed ESI-MS titration experiments on six high-affinity Cu(II)/peptide systems which we previously characterized in a comprehensive fashion.

Figure 2

Figure 2. Theoretical dissociation curves of Cu(II)/peptide 1:1 complexes at pH 7.4.

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We started with serial dilution experiments on a standard ESI-MS instrument in order to obtain apparent affinity constants serving for quantitative illustration of the scale of the systematic error generated by ESI-MS. This was followed by pH–jump kinetic experiments aimed to model events occurring in the evaporating spray droplets and by additional competition-based affinity studies performed on an ESI-MS instrument dedicated for studies of noncovalent interactions. All these experiments yielded a clear and unequivocal view that ESI-MS should not be used for quantitation of Cu(II)/peptide complexes.

Experimental Section

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l-Histidine and ammonium acetate were purchased from Sigma. All peptides, except for GGH purchased from Sigma, were synthesized in-house with standard Fmoc solid phase synthesis, as described before. (62) Crude synthesis products were purified with RP-HPLC on ACE C18-300 column 250 × 8 mm with a rising gradient of acetonitrile in water with 0.1% TFA.
ESI-MS spectra were recorded on a Premier ESI-QToF spectrometer (Waters). All measurements were performed in the positive ion mode. The source temperature 80 °C was used for a complete desolvation of the peptide ions. The cone voltage was 10 V for shorter peptides and 30 V for Aβ4–16. The transmission of the ions was optimized on the quadrupole for the required mass range (m/z 200 to 1000 for shorter peptides, m/z 300 to 1500 for Aβ4–16). Mass spectra were accumulated over 2 to 3 min to improve the signal-to-noise ratio. The sample flow was 20 μL/min. The 100 μM Cu(II)-peptide solutions in 20 mM ammonium acetate at pH 7.4 were used for serial dilutions of the whole complex with the same ammonium acetate. For quantitation, all peaks corresponding to each charged state were integrated for each peptide. Ratios of free peptide to Cu(II)/peptide complex were extracted from the integration of MS intensities with the assumption of similar ionization efficacy. For each dilution the Kd was calculated according to eq 1, followed by the averaging as required (see the Results for details).
(1)
The free Cu2+ ions could not be detected directly, due to low m/z and instead were calculated from the mass balance (free Cu2+ assumed to be equal to free peptide).
Isotopic distributions were calculated using built-in MassLynx Isotope model software (Waters).
Competition experiments with histidine were carried out on Q Exactive UHMR Hybrid Quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific). Samples were prepared by dissolving peptides in 50 mM ammonium acetate pH 7.4 in 100 nM concentration. To each sample a mixture of 100 nM CuCl2 and differing concentrations of histidine were added. Measurements started with an 1 h delay to allow for reaching equilibrium. Samples were introduced into the mass spectrometer with a syringe pump using 10 μL/min flow rate, by electrospray ionization using positive mode in HESI source. MS measurements were conducted under the following settings: desolvation voltage: 20 V, capillary temperature 320 °C, detector m/z optimization: low m/z; ion transfer optimization to low m/z. The RF applied throughout the instrument were set to 150 Vp-p for injection flatapole, 300 Vp-p for bent flatapole, 250 for transfer multipole and HCD cell, and 2300 for C-trap. The ions transfer optics was to 5 V for injection flatapole, 4 V for intel flatapole, 2 V for bent flatapole and 0 V for transfer multipole. Integration of the resulting peaks was achieved with built-in software FreeStyle 1.4 (Thermo Scientific).
The stopped-flow SFM-300 (BioLogic) instrument was used to measure the rates of dissociation of Cu(II)–GGH and Cu(II)–hepc6 complexes upon acidification. The kinetic runs were observed with a diode-array detector (TIDAS S 500 K, J&M Analitik AG), with the spectra recorded in the 400–900 nm wavelength range at 1.5 ms intervals. Reactions were performed in a 1 cm path length cuvette at 25 °C. The dead time of the instrument was 2 ms at total flow rate 15 mL/min.
The pH–jump experiments were carried out for solutions of 2 mM peptide and 1.8 mM CuCl2 in 50 mM ammonium acetate at pH = 7.4 mixed with equal volumes of 45 mM, 0.25 M, or 2.5 M acetic acid, which resulted in sample acidification to pH 5, 4, or 3, respectively. The dilution during the mixing yielded final cuvette concentrations of 1 mM peptide, 0.9 mM Cu(II), and 22.5 mM, 125 mM and 1.25 M acetic acid, respectively. The solutions were freshly prepared and degassed before each series of reactions. The final pH value was measured in the samples collected after each experiment. To show the differences in absorbance signal before the first recorded time point, the dilution of the Cu(II)–peptide complex was measured as a control (shown as t = 0 s). In all cases, the peptides were in a slight excess over Cu(II) to avoid Cu(OH)2 precipitation.

Results

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ESI-MS Attempts at Determining CK

To test the pseudophysiological ESI-MS conditions for the binding constant determination we employed two different mass spectrometers and two different techniques. Serial dilution studies of Cu–peptide complexes were performed on a standard ESI-MS instrument, while histidine competition experiments were performed on a sensitive instrument dedicated for studies of noncovalent complexes.
In the first approach, we performed measurements on several synthetic peptides with well-established Cu(II) binding properties. Equimolar Cu(II) peptide mixtures (100 μM) were dissolved in 20 mM ammonium acetate at pH 7.4. The dissociation of complexes was monitored by serial dilutions of both reagents. Table 1 provides the Kd values obtained according to eq 1 for each data point and averaged. These values were compared to the literature CK7.4 values. (6,21,63−65) Very large discrepancies were observed, ranging from five to eight log units.
The ESI-MS spectra are presented in Figures S1–S6. As seen in these figures, substantial amounts of unbound peptides were observed in all cases at all tested concentrations. For example, ca. 20% free peptide was detected throughout the dilution experiment for hepc6. This degree of Cu(hepc6) complex dissociation at pH 7.4 should not occur above a complex concentration of 9 fM (Figure 2). (63) Further experiments were undertaken to explain the sources of this discrepancy.
For the peptides forming the weakest (GGH) and the strongest complexes (hepc6), different Cu(II)/peptide ratios were analyzed (Figure S7 and S8, respectively). The increasing Cu(II) amounts should eventually saturate the peptide, eliminating the apopeptide signals. This did not happen, even at the highest Cu(II) excess that could be achieved due to solubility, more than hundred-fold for GGH (Figure S7). As presented in Figure S9, increasing the Cu/peptide ratio lowered the apparent Kd value, contrary to what should be expected according to eq 1.
We also analyzed the spectra for the signs of Cu(II) reduction to Cu(I) as a possible source of Kd value deviation. This effect would manifest itself qualitatively in the ESI-MS spectra as a m/z shift of +1 for the molecular ions containing Cu(I), because the positive charge decrease on the copper ion would have to be compensated by an additional H+ ion attached elsewhere. This, in turn, would affect the peak proportions in the ionic manifold. This effect was analyzed by comparing the peak maximum intensities with respect to the monoisotopic (highest) peak. Indeed, very slight increases of the second and fourth peaks were observed in most peptides, consistent with the +1 mass shift, but the effect was small, not exceeding a few percent of the monoisotopic peak (Figure S10). Thus, some Cu(II) reduction to Cu(I) could have occurred in our experiments, but its contribution to Kd was minimal to negligible.

Competition Experiments with Histidine

Having demonstrated that serial dilution experiments on a standard ESI-MS instrument inadvertently yield artifacts, we checked whether competition experiments could provide a better result. For that we used a state-of-the-art Q-Exactive Ultra-High Mass Range mass spectrometer designed specially to preserve native noncovalent bonds. The initial experiment in 50 mM ammonium acetate, pH 7.4, proved that no artifactual complex dissociation occurred for the CuAβ4–16 complex at the 100 pM concentration, near the instrument’s detection limit (Figure S11). According to Figure 2, this dilution was still 2 orders of magnitude away from the range at which serial dilution could be applied but gave promise for competition experiments. In this approach, 100 nM peptides Aβ4–16 and hepc6 mixed with 100 nM Cu(II) were titrated with histidine, which served as a weak competitor. With full knowledge of the protonation states and stability constants for each complex species in solution (Tables S1 and S2) we were able to calculate the theoretical binding isotherms indicating a feasibility of such approach. The experimental curves were unfortunately different (Figure 3).

Figure 3

Figure 3. Experimental and theoretical binding isotherms for Cu(II)/Aβ4-16 (A) and Cu(II)/hepc6 (B) 100 nM 1:1 complexes titrated with histidine as a weak competitor. Dotted lines mark concentration at which 50% of the peptide is occupied with Cu(II) ion.

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Calculating binding constants from competition experiments where the competitor forms both 1:1 and 2:1 complexes is not an easy task in general, and competition may produce errors that are difficult to account for, (66) but we were able to circumvent the calculation problem by extracting the histidine concentration at which 50% of peptides were bound to Cu(II) from Figure 3. These values were 24.9 μM His for Aβ4–16 and 93.3 μM His for hepc6. With these values and the knowledge of all protonation constants of histidine and stability constants for Cu(II)/histidine complexes (67) we could calculate the binding constants using the competitivity index (CI) approach. (68,69) The CI for a binary metal/ligand system is defined as the logarithm of the conditional stability constant of MZ (the metal complex of a theoretical molecule Z), such that Σijk([MiHjLk]) = [MZ], at given overall component concentrations, where M is a metal ion, H is hydrogen, and L is the metal binding ligand. In other words, the apparent stability constant of MZ, in units of M–1, reflects the overall metal ion binding ability of all other molecules in equilibrium. CI is equivalent to log CK7.4 when there is only one complex species under given conditions but has a broader relevance if more than one species coexist in solution. In such case CI represents the metal binding properties of the whole ensemble of complex species. In the studied system L is histidine and Z is the peptide. The data obtained through these calculations are gathered in Table 2, and raw spectra are shown in Figures S12 and S13.
Table 2. Comparison of Conditional Cu(II) Binding Constants Obtained with Competition Experiments with Histidine of the Studied Peptides at pH = 7.4 (CK7.4), Compared to These Constants Determined Previously by Other Methods and Publisheda
peptide sequence (name)published log CK7.4log Kd from His competition ESI-MS experiments in this studylog ratio log Kd (ESI-MS) - log CK7.4 (published)range of concentrations of histidine in this study (μM)
DTHFPI-NH2 (hepc6)14.66b13.98–0.681000-10
FRHDSGYEVHHQK-NH2 (Aβ4–16)13.53c12.84–0.69500-1
a

Logarithmic values are given for better clarity. The published log CK7.4 values were determined by potentiometry and corroborated by spectroscopic methods.

b

Reference (63).

c

Reference (6).

Modeling the pH Drop in Electrospray Droplets Using pH–jump Stopped Flow

The formation of Cu(II)–peptide complexes in general and ATCUN complexes in particular is very strongly pH-dependent. (18) This is illustrated in Figure 4 for hepc6 and GGH and in Figure S14 for other studied peptides. These diagrams indicate that the significant coexistence of Cu(II)-bound and unbound peptides occurs typically in the pH range between 3 and 5. The presence of unbound peptides in all serial dilution ESI-MS experiments could thus be a simple consequence of acidification of sample droplets containing ammonium acetate during their evolution in electrospray. (52)

Figure 4

Figure 4. pH-dependent species distribution of Cu(II) complexes of hepc6 (top) and GGH (bottom) calculated for 1 mM peptides and 0.8 mM Cu(II) based on potentiometric data from ref (65) and ref (21), respectively.

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Figure 5 provides the correlation diagram indicating what pH values in electrospray droplets would explain the apparent loss of Cu(II) affinity if droplet acidification were the only source of the observed log Kd decrease in serial dilution experiments. Although all values are within the buffering range of acetate, their spread is rather random and covers about one pH unit, despite using the same experimental conditions in all respective experiments. Therefore, the acidification, while plausible, is not the only source of the studied effect and the actual Kd values cannot be simply recovered from ESI-MS data by adjusting them for a hypothetical electrospray pH value.

Figure 5

Figure 5. Prediction of pH values in electrospray stipulated by the literature log CK values compared with the values calculated from serial dilution ESI-MS (data from Table 1). Solid curves represent the pH dependence of literature log CK values for individual peptides. Circles mark the ESI-MS-derived log Kd values ± SD placed along these curves to indicate the pH of spray droplets expected if acidification were the sole source of the observed Kd decrease. Gray area marks the buffering zone of ammonium acetate (pH 4.75 ± 1).

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Cu(II) complexes are usually considered to equilibrate rapidly, but our recent study demonstrated that Cu(II) complexes of ATCUN peptides are formed in a stepwise manner, with the lifetime of most stable intermediate species around several hundred milliseconds. (20) A typical time of electrospray formation is ≤10 ms. (37,38) These facts prompted us to investigate whether the complexes had enough time to reach the new pH-dependent equilibrium within the time of droplet evolution preceding the gas phase transition. To answer this question, we performed stopped-flow pH–jump experiments. The samples containing Cu(II)–hepc6 and Cu(II)–GGH complexes in ammonium acetate at pH 7.4 were mixed with acetic acid solutions of concentrations adjusted to reach pH 5, 4, or 3 after the sample mixing. The evolution of the systems was monitored using the visible absorption spectra, as shown in Figure 6 for Cu(II)–hepc6. The data for Cu(II)–GGH are provided in Figure S15.

Figure 6

Figure 6. Stopped-flow spectra of Cu(II)-hepc6 in 20 mM ammonium acetate initially at pH 7.4, subjected to rapid pH decrease by mixing with concentrated acetic acid. Color-coded spectra were recorded every 1.5 ms with the instrument dead time of 2 ms. (A) First 11 ms of the reaction and (B) all spectra recorded until equilibrium was reached. The band at 525 nm represents the fully formed (4N) ATCUN complex, stable at pH 7.4, and the band at 750 nm represents the intermediate (2N) complex. (20)

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The traces at 525 nm, corresponding to the decomposition of the 4N ATCUN complex, are presented in Figure 7 for Cu(II)–hepc6 and in Figure S16 for Cu(II)–GGH. The extent of dissociation of 4N complexes after 10 ms at pH 5, 4, and 3 was measured by following the decay of their absorption peaks at 525 nm. Due to significant noise of the absorption signal the averaged signal intensities at 0 and 10 ms were obtained by fitting a first order kinetic function to the absorption data. The overall fits and expanded regions of interest (0 to 30 ms) are presented for clarity. As seen in these figures, the inertness of ATCUN complexes to acid-catalyzed dissociation in the 10 ms time window is significant (except for Cu(II)–GGH at pH 3) and must be taken into account. Table S3 presents the results of a simulation of relative abundances of apopeptides and Cu(II) complexes that should occur if the ESI-MS signals depended only on droplet acidification and the resulting complex dissociation after 10 ms. This analysis indicates that the abundance of hepc6 apopeptide in ESI-MS spectra is consistent with pH the droplet drop to slightly above 3, while the abundance of GGH apopeptide appears to correspond with the final droplet pH clearly above 5. Both cannot be true for experiments performed under identical conditions. Hence, acidification and complex decay kinetics do not explain the pattern of free and complexed peptides observed in ESI-MS.

Figure 7

Figure 7. Kinetic traces at 525 nm for pH–jump experiments on Cu(II)-hepc6 presented in Figure 6 for pH 3 (A), 4 (B), and 5 (C). Left: full traces recorded over 1.5 s and 1st order kinetic fits (red lines). Right: the first 30 ms of traces. Vertical lines mark the 0 ms (reaction start) and the 10 ms time points.

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The application of the above methodology to decipher the mechanism responsible for the deviation in competition experiments is not possible because of the overlap of the pH and desolvation effects exerted simultaneously on the peptide and histidine complexes. The droplet acidification and/or complex unfolding processes are less significant in the mild desolvation conditions of the respective instrument, as evidenced by the control experiment. The theoretical speciation for Cu(Aβ4–16) at the 100 pM concentration suggests that the pH drop may be actually very low or none (Figure S11). In accord, the attempt to assign a pH drop to competition experiments proved futile, as presented in Figure 8. Therefore, the systematic error embedded in these experiments is likely due to complex desolvation phenomena, as discussed below.

Figure 8

Figure 8. Experimental and theoretical binding isotherms of Cu/Aβ4-16 (A) and Cu/hepc6 (B) 1:1 complexes titrated with histidine as weak competitor.

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Discussion

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The experimental results presented in Figure 3 and Figures S1–S6 and S12–S13 showed that the proportions of free ATCUN peptides and their complexes in ammonium acetate solutions at pH 7.4, obtained by ESI-MS, do not match the expectations based on CK7.4 values obtained by other techniques (Tables 1 and 2). This cannot be assigned to a nonproportionate efficacy of ionization of respective species, a well-known phenomenon in ESI-MS, (30) because the discrepancy is far too high.
In the case of histidine competition experiments, a disproportional ionization would shift the binding curve but should not yield a steeper transition from free to complexed peptide. Doing so would indicate that ionization efficiency difference reverses in changing concentrations of the competitor. Interestingly, at low histidine concentrations the apparent Cu(II) binding by the peptides is slightly enhanced beyond the expected value. This could be caused by the fact that during shrinking of the droplet, when concentrations rise, His escapes the droplets earlier. This would shift the equilibrium in favor of the peptide. No such effect is observed, however, at higher histidine concentrations.
In the case of serial dilutions, the artifactual surplus of free peptides was not alleviated even at a high Cu2+ excess (Figures S7–S9). The analysis of relative peak intensities excluded the Cu(II) reduction to Cu(I) as a significant source of this discrepancy (Figure S10). The significant amounts of apopeptides in the spectra resembled the Cu2+/peptide equilibria under the acidic conditions (Figures 4 and S14), which appeared to correlate with the noted acidification of ammonium acetate solutions upon the electrospray droplet formation. (52) Quantitative analysis demonstrated, however, that the apparent pH values derived from ESI-MS data are spread randomly over nearly one pH unit (5.0–5.7, Figure 5), effectively precluding a quantitative use of such data. Additionally, this apparent pH shift was less than expected for droplet acidification according to Konermann (around 4.75). (52) Looking for a reason, we turned to pH–jump kinetic experiments, guided by our recent discovery of intermediate steps in the process of ATCUN complex formation, which could overlap with the lifetime of electrospray droplets. (20,41) These experiments, presented in Figures 6, 7, S15, and S16, revealed that the studied complexes are far too inert to undergo a significant decomposition within the 10 ms time window facing the relevant extent of acidification. Therefore, the main reason for the complex dissociation was in this case an inadequate ESI source or its parameters. Tuning of voltage, gas flow, or injection flow did not yield considerable success. However, changing the spectrometer to the one dedicated to intact noncovalent bonds yielded much more accurate results. The apparent binding constants were much closer to expected values derived from solution studies but were still significantly underestimated.
We propose that the clue for the observed effect is provided by thermodynamics of Cu(II) complex formation. The ITC investigation of DAHK, an ATCUN peptide model of human serum albumin, demonstrated that the Cu(DAHK) complex at neutral pH is stabilized solely by entropic contribution. (50) At this pH the enthalpic contributions such as Coulombic attraction are canceled out by the cost of deprotonations of peptide nitrogens some 8 orders of magnitude below its Ka. (70) The main contributor to complex stability is believed to be a release of water molecules from hydration shells of Cu(II) and the peptide, as well as deprotonation. Stable 4-nitrogen–Cu(II) complex leaves much less peptide and metal exposed to the solvent; thus, the entropic factor comes from the combination of folding and desolvation. A similar feature was observed for Cu(II) binding to metalloproteins. (51,71) Therefore, in accordance with the data presented above, the removal of water from the second coordination sphere of a Cu(II)–ATCUN complex will likely lead to the equilibrium shift and possible subsequent complex decomposition upon entry of the molecules into the gas phase. It was previously established that thermodynamic changes occur during transition to the gas phase and entropically favored forces become destabilized. (28) Additionally, acid-dissociated Cu2+ ions may recombine with the peptide upon its transition to the gas phase, additionally confounding the observations. (72) From this perspective, one can state that neither ESI-MS nor other mass spectrometry techniques should be applied in quantitative studies of Cu(II)–peptide complexes. If such experiments are performed, the resulting binding constant will always be lower than the real one, but to an extent impossible to predict without independent data obtained by a different method. Therefore, mass spectrometry data for Cu(II)/peptide binding constants should be regarded as “greater than x”, always with an assumption that the in-solution binding constant is higher than that measured. On the other hand, enthalpic contributions are significant or decisive for the stability of other metal chelates in water, (73,74) and hence, metal complex formation can be studied by MS upon maintaining the utmost scrutiny and validating the results by independent techniques whenever possible.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jasms.1c00206.

  • Serial dilution data (Figures S1–S6), Cu2+ titrations (Figures S7 and S8) and derived apparent Kd (Figure S9), isotopic distributions for detection of Cu2+ reduction (Figure S10), calculated pH distribution of Cu(II)Aβ4-16 (Figure S11), raw spectra for histidine competition (Figures S12 and S13), literature log β values for Cu(II) complexes of hepc6 and Aβ4-16 (Tables S1 and S2), species distributions for Cu(II) complexes of MNH-NH2, Aβ4-9, Aβ4-16, and MDH-NH2 (Figure S14), stopped-flow spectra (Figure S15), kinetic traces (Figure S16), and recalculation of apparent spray pH (Table S3) (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • Dawid Płonka - Institute of Biochemistry and Biophysics, Polish Academy of Sciences Pawińskiego 5A, 02-106 Warsaw, Poland;  Orcidhttps://orcid.org/0000-0002-4076-9231
    • Radosław Kotuniak - Institute of Biochemistry and Biophysics, Polish Academy of Sciences Pawińskiego 5A, 02-106 Warsaw, Poland
    • Katarzyna Dąbrowska - Institute of Biochemistry and Biophysics, Polish Academy of Sciences Pawińskiego 5A, 02-106 Warsaw, Poland
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This work was supported by National Science Center (Poland) PRELUDIUM projects no. 2018/31/N/ST4/01259 (D.P) and 2018/31/N/ST5/02556 (R.K.) The equipment used was sponsored, in part, by the Centre for Preclinical Research and Technology (CePT), a project cosponsored by the European Regional Development Fund and Innovative Economy, The National Cohesion Strategy of Poland. All measurements on Q Exactive UHMR Hybrid Quadrupole-Orbitrap mass spectrometer were done in the Mass Spectrometry Laboratory of Institute of Biochemistry and Biophysics, Polish Academy of Sciences in terms of demonstration laboratory by Thermo Scientific.

References

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This article references 74 other publications.

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    The tachykinin neuropeptide, neurokinin B (NKB), belongs to a family of peptides having diverse roles in the brain. NKB, along with several other tachykinins, has been identified as a copper-binding peptide, however the physiol. relevance of the binding is unclear. Previously, NKB was shown to limit the ability of copper to enter astrocytes and disrupt calcium homeostasis and it was thought that the peptide was sequestering the metal extracellularly. Here we use a fluorescein-labeled NKB peptide (F-NKB) to show that NKB is not retained extracellularly, but is endocytosed within 10-20 min after addn. to the cell media. The endocytosis is not inhibited when NKB is delivered as a copper-complex, [CuII(F-NKB)2]. Endocytosis of NKB can increase intracellular copper. Comparison to cells cultured in copper-free buffer indicated that apo-NKB can facilitate uptake of copper found in normal culture media. To achieve this NKB must compete with a variety of copper proteins, and we show that NKB can successfully compete with copper-binding peptides derived from the prion protein, itself assocd. with Cu(II) and Zn(II) metab. We suggest a mechanism of receptor mediated endocytosis to account for the observations.
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    Chemical Research in Toxicology (1997), 10 (8), 915-921CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
    The potential of Ni(II) and Cu(II) complexes with Arg-Thr-His-Gly-Gln-Ser-His-Tyr-Arg-Arg-Arg-His-Cys-Ser-Arg-amide (HP21-15), a peptide modeling the N-terminal amino acid sequence of human protamine HP2, to mediate oxidative DNA damage was studied by measurements of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) generation from 2'-deoxyguanosine (dG) and calf thymus DNA and by formation of double-strand breaks in calf thymus DNA. The concns. of reagents were 0.1 mM dG and the metal-HP21-15 complex, 1 mM H2O2, 1.5 mM DNA (per phosphate group), 100 mM phosphate buffer, pH 7.4, ambient O2. Samples were incubated at 37° for 16-24 h. The Cu(II)-HP21-15 complex was found to be an effective promoter of the formation of 8-oxo-dG from both dG and DNA with ambient O2 (approx. 13- and 3-fold increase vs. the oxidant alone, resp.) and H2O2 (approx. 25-fold increase in either case). The Ni(II)-HP21-15 complex was ineffective with O2 vs. 8-oxo-dG prodn. from both substrates but markedly enhanced the attack of H2O2 on dG and DNA (approx. 5-fold increase of 8-oxo-dG prodn. in either case). Both Cu(II)- and Ni(II)-HP21-15 equally promoted double-strand scission by H2O2 in calf thymus DNA. The promotion by the complexes of dG and DNA oxidn. with H2O2 was accompanied by oxidative damage to the complexes themselves, consisting of decreasing contents of their His (to approx. 50% of control in either complex) and esp. Tyr (down to 48% of control in Cu(II)- and 19% in Ni(II)-HP21-15) residues, as well as appearance of aspartic acid, the known oxidn. product of His residues in peptides (up to 22% vs. Gly for Cu(II)- and 10% for Ni(II)-HP21-15). The above results provide a novel chem. mechanism of Cu(II) and Ni(II) toxicity and may have wide implications for reproductive and transgenerational effects of metal exposure.
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    Liang, R.; Senturker, S.; Shi, X.; Bal, W.; Dizdaroglu, M.; Kasprzak, K. S. Effects of Ni(II) and Cu(II) on DNA interaction with the N-terminal sequence of human protamine P2: Enhancement of binding and mediation of oxidative DNA strand scission and base damage. Carcinogenesis 1999, 20, 893– 898,  DOI: 10.1093/carcin/20.5.893
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    Carcinogenesis (1999), 20 (5), 893-898CODEN: CRNGDP; ISSN:0143-3334. (Oxford University Press)
    Epidemiol. evidence suggests that certain paternal exposures to metals may increase the risk of cancer in the progeny. This effect may be assocd. with promutagenic damage to the sperm DNA. The latter is packed with protamines which might sequester carcinogenic metals and moderate the damage. Human protamine P2 has an amino acid motif at its N-terminus that can serve as a heavy metal trap, esp. for Ni(II) and Cu(II). We have synthesized a pentadecapeptide modeling this motif, Arg-Thr-His-Gly-Gln-Ser-His-Tyr-Arg-Arg-Arg-His-Cys-Ser-Arg-amide (HP21-15) and described its complexes with Ni(II) and Cu(II), including their capacity to mediate oxidative DNA degrdn. (1997). In the present study, effects of HP21-15 on Ni(II)- and Cu(II)-mediated DNA oxidn. by H2O2 at pH 7.4 were investigated in more detail using the circular plasmid pUC19 DNA as a target, and the single/double-strand breaks and prodn. of oxidized DNA bases, as end points. Ni(II) alone was found to promote oxidative DNA strand scission (mostly single strand breaks) and base damage, while Cu(II) alone produced the same effects, but to a much greater extent. Both metals were relatively more damaging to the pyrimidine bases than to purine bases. HP21-15 tended to increase the Ni(II)/H2O2-induced DNA breakage. In sharp contrast, the destruction of DNA strands by Cu(II)/H2O2 was almost completely prevented by HP21-15. The effect of HP21-15 on the oxidative DNA base damage varied from a limited enhancement (5-hydroxyhydantoin and thymine glycol) to slight suppression (5-hydroxycytosine, 5-hydroxyuracil, 8-oxoguanine, 8-oxoadenine, 2-hydroxyadenine, fapyguanine and fapyadenine) toward Ni(II)/H2O2. HP21-15 strongly suppressed the oxidative activity of Cu(II)/H2O2 in regard to all bases in DNA. Consistently with the above, the ESR/spin trap measurements revealed greater and more persistent generation of OH· and O2-•-like oxidants from H2O2 by the Ni(II)-HP21-15 complex than by the Cu(II)-HP21-15 complex (no O2-•-was detected). Both complexes were also found to bind to DNA more strongly than HP21-15 alone. The results indicate that protamine P2 is capable of binding Ni(II) and Cu(II) and, in this way, attenuating the mediation of oxidative DNA damage by Cu(II), but not Ni(II). The effects found may be mechanistically involved in the reproductive toxicity and carcinogenicity of metals.
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    Conklin, S. E.; Bridgman, E. C.; Su, Q.; Riggs-Gelasco, P.; Haas, K. L.; Franz, K. J. Specific Histidine Residues Confer Histatin Peptides with Copper-Dependent Activity against Candida albicans. Biochemistry 2017, 56, 4244– 4255,  DOI: 10.1021/acs.biochem.7b00348
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    Specific Histidine Residues Confer Histatin Peptides with Copper-Dependent Activity against Candida albicans
    Conklin, Steven E.; Bridgman, Emma C.; Su, Qiang; Riggs-Gelasco, Pamela; Haas, Kathryn L.; Franz, Katherine J.
    Biochemistry (2017), 56 (32), 4244-4255CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The histidine-rich salivary peptides of the histatin family are known to bind copper (Cu) and other metal ions in vitro, but the details of these interactions are poorly understood and their implications on in vivo antifungal activity have not been established. Here, we show that availability of Cu during exposure of Candida albicans to histatin-5 (Hist-5) modulates its antifungal activity. Antifungal susceptibility testing revealed that cotreatment of Hist-5 with Cu improved the EC50 from ∼5 μM to ∼1 μM, whereas cotreatment with a high-affinity Cu-specific chelator abrogated antifungal activity. Spectrophotometric titrns. revealed two previously unrecognized Cu(I) binding sites with apparent Kd values at pH 7.4-20 nM, and confirmed a high-affinity Cu(II) binding site at the Hist-5 N-terminus with apparent Kd ∼ 8 pM. Evaluation of a series of His-to-Ala peptides contg. the first 12 residues of Hist-5 identified adjacent His residues (bis-His) as crit. anchors for Cu(I) binding, and the presence of a third ligand was revealed by X-ray absorption spectroscopy (XAS). On their own, the truncated peptides were ineffective at inhibiting growth of C. albicans, but treatment with supplemental Cu resulted in EC50 values down to ∼5 μM, approaching that of full-length Hist-5. The efficacy of the peptides depended on an intact bis-His site and correlated with Cu(I) affinity. Together, these results establish new structure-function relationships linking specific histidine residues with Cu-binding affinity and antifungal activity, and provide further evidence for the involvement of metals in modulating the biol. activity of these antifungal peptides.
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  12. 12
    Frączyk, T. Cu(II)-Binding N-Terminal Sequences of Human Proteins. Chem. Biodiversity 2021, 18, e2100043  DOI: 10.1002/cbdv.202100043
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    12
    Cu(II)-Binding N-Terminal Sequences of Human Proteins
    Fraczyk, Tomasz
    Chemistry & Biodiversity (2021), 18 (4), e2100043CODEN: CBHIAM; ISSN:1612-1872. (Verlag Helvetica Chimica Acta)
    Proteins anchor copper(II) ions mainly by imidazole from histidine residues located in different positions in the primary protein structures. However, the motifs with histidine in the first three N-terminal positions (His1, His2, and His3) show unique Cu(II)-binding properties, such as availability from the surface of the protein, high flexibility, and high Cu(II) exchangeability with other ligands. It makes such sequences beneficial for the fast exchange of Cu(II) between ligands. Furthermore, sequences with His1 and His2, thus, non-satg. the Cu(II) coordination sphere, are redox-active and may play a role in Cu(II) redn. to Cu(I). All human protein sequences deposited in UniProt Knowledgebase were browsed for those contg. His1, His2, or His3. Proteolytically modified sequences (with the removal of a propeptide or Met residue) were taken for the anal. Finally, the sequences were sorted out according to the subcellular localization of the proteins to match the resp. sequences with the probability of interaction with divalent copper.
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  13. 13
    Sokolowska, M.; Krezel, A.; Dyba, M.; Szewczuk, Z.; Bal, W. Short peptides are not reliable models of thermodynamic and kinetic properties of the N-terminal metal binding site in serum albumin. Eur. J. Biochem. 2002, 269, 1323– 1331,  DOI: 10.1046/j.1432-1033.2002.02772.x
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    13
    Short peptides are not reliable models of thermodynamic and kinetic properties of the N-terminal metal binding site in serum albumin
    Sokolowska, Magdalena; Krezel, Artur; Dyba, Marcin; Szewczuk, Zbigniew; Bal, Wojciech
    European Journal of Biochemistry (2002), 269 (4), 1323-1331CODEN: EJBCAI; ISSN:0014-2956. (Blackwell Publishing Ltd.)
    A comparative study of thermodn. and kinetic aspects of Cu(II) and Ni(II) binding at the N-terminal binding site of human and bovine serum albumins (HSA and BSA, resp.) and short peptide analogs was performed using potentiometry and spectroscopic techniques. It was found that while qual. aspects of interaction (spectra and structures of complexes, order of reactions) could be reproduced, the quant. parameters (stability and rate consts.) could not. The N-terminal site in HSA is much more similar to BSA than to short peptides reproducing the HSA sequence. A very strong influence of phosphate ions on the kinetics of Ni(II) interaction was found. This study demonstrates the limitations of short peptide modeling of Cu(II) and Ni(II) transport by albumins.
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  14. 14
    Haas, K. L.; Putterman, A. B.; White, D. R.; Thiele, D. J.; Franz, K. J. Model peptides provide new insights into the role of histidine residues as potential ligands in human cellular copper acquisition via Ctr1. J. Am. Chem. Soc. 2011, 133, 4427– 4437,  DOI: 10.1021/ja108890c
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    14
    Model Peptides Provide New Insights into the Role of Histidine Residues as Potential Ligands in Human Cellular Copper Acquisition via Ctr1
    Haas, Kathryn L.; Putterman, Allison B.; White, Daniel R.; Thiele, Dennis J.; Franz, Katherine J.
    Journal of the American Chemical Society (2011), 133 (12), 4427-4437CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Cellular acquisition of copper in eukaryotes is primarily accomplished through the Ctr family of copper transport proteins. In both humans and yeast, methionine-rich "Mets" motifs in the amino-terminal extracellular domain of Ctr1 are thought to be responsible for recruitment of copper at the cell surface. Unlike yeast, mammalian Ctr1 also contains extracellular histidine-rich motifs, although a role for these regions in copper uptake has not been explored in detail. Herein, synthetic model peptides contg. the first 14 residues of the extracellular domain of human Ctr1 (MDHSHHMGMSYMDS) have been prepd. and evaluated for their apparent binding affinity to both Cu(I) and Cu(II). These studies reveal a high affinity Cu(II) binding site (log K = 11.0 ± 0.3 at pH 7.4) at the amino-terminus of the peptide as well as a high affinity Cu(I) site (log K = 10.2 ± 0.2 at pH 7.4) that utilizes adjacent HH residues along with an addnl. His or Met ligand. These model studies suggest that the histidine domains may play a direct role in copper acquisition from serum copper-binding proteins and in facilitating the redn. of Cu(II) to the active Ctr1 substrate, Cu(I). We tested this hypothesis by expressing a Ctr1 mutant lacking only extracellular histidine residues in Ctr1-knockout mouse embryonic fibroblasts. Results from live cell studies support the hypothesis that extracellular amino-terminal His residues directly participate in the copper transport function of Ctr1.
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  15. 15
    Stefaniak, E.; Płonka, D.; Drew, S. C.; Bossak-Ahmad, K.; Haas, K. L.; Pushie, M. J.; Faller, P.; Wezynfeld, N. E.; Bal, W. The N-terminal 14-mer model peptide of human Ctr1 can collect Cu(ii) from albumin. Implications for copper uptake by Ctr1. Metallomics 2018, 10, 1723– 1727,  DOI: 10.1039/C8MT00274F
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    15
    The N-terminal 14-mer model peptide of human Ctr1 can collect Cu(II) from albumin. Implications for copper uptake by Ctr1
    Stefaniak, Ewelina; Plonka, Dawid; Drew, Simon C.; Bossak-Ahmad, Karolina; Haas, Kathryn L.; Pushie, M. Jake; Faller, Peter; Wezynfeld, Nina E.; Bal, Wojciech
    Metallomics (2018), 10 (12), 1723-1727CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)
    Human cells acquire copper primarily via the copper transporter 1 protein, hCtr1. We demonstrate that at extracellular pH 7.4 CuII is bound to the model peptide hCtr11-14via an ATCUN motif and such complexes are strong enough to collect CuII from albumin, supporting the potential physiol. role of CuII binding to hCtr1.
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    Arena, G.; La Mendola, D.; Pappalardo, G.; Sóvágó, I.; Rizzarelli, E. Interactions of Cu2+ with prion family peptide fragments: Considerations on affinity, speciation and coordination. Coord. Chem. Rev. 2012, 256, 2202– 2218,  DOI: 10.1016/j.ccr.2012.03.038
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    16
    Interactions of Cu2+ with prion family peptide fragments: Considerations on affinity, speciation and coordination
    Arena, Giuseppe; La Mendola, Diego; Pappalardo, Giuseppe; Sovago, Imre; Rizzarelli, Enrico
    Coordination Chemistry Reviews (2012), 256 (19-20), 2202-2218CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)
    A review. The review describes the stability and the coordination modes of Cu2+ complexes with different regions of N-terminus prion proteins. The structural features of the different metal species are correlated both with the Cu2+-driven redox properties and with the conformational changes induced by the Cu2+ in the different metal binding regions of the protein. The formation of mixed metal complexes is also discussed. We emphasize that binding features should be discussed by referring to the species that actually forms under specific conditions (pH, buffer, etc.) rather than to the 'binding site'; correlating properties with the structures of the so called 'binding sites' may lead to misinterpretation of the exptl. results, since a 'binding site' often corresponds to a mixt. of species. We also highlight that ignoring species that form with ligands other than the prion peptide (e.g. the buffer) may lead to underestimating their role in crucial processes (e.g. redox activity).
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  17. 17
    Zawisza, I.; Rózga, M.; Bal, W. Affinity of copper and zinc ions to proteins and peptides related to neurodegenerative conditions (Aβ, APP, α-synuclein, PrP). Coord. Chem. Rev. 2012, 256, 2297– 2307,  DOI: 10.1016/j.ccr.2012.03.012
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    17
    Affinity of copper and zinc ions to proteins and peptides related to neurodegenerative conditions (Aβ, APP, α-synuclein, PrP)
    Zawisza, Izabela; Rozga, Malgorzata; Bal, Wojciech
    Coordination Chemistry Reviews (2012), 256 (19-20), 2297-2307CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)
    A review. The review describes the state of the art in the field of stability const. detn. for Cu(II), Cu(I) and Zn(II) complexes of proteins and peptides involved in neurodegenerative diseases, α-synuclein (aS), prion protein (PrP), amyloid precursor protein (APP) and amyloid β peptides (Aβ). The methodologies and results are critically analyzed and recommendations are formulated about possible systematic errors in these studies. The possibility of formation of ternary complexes with titrn. competitors is discussed.
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  18. 18
    Gonzalez, P.; Bossak, K.; Stefaniak, E.; Hureau, C.; Raibaut, L.; Bal, W.; Faller, P. N-Terminal Cu-Binding Motifs (Xxx-Zzz-His, Xxx-His) and Their Derivatives: Chemistry, Biology and Medicinal Applications. Chem. - Eur. J. 2018, 24, 8029– 8041,  DOI: 10.1002/chem.201705398
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    18
    N-terminal Cu-binding motifs (Xxx-Zzz-His, Xxx-His) and their derivatives: Chemistry, biology and medicinal applications
    Gonzalez, Paulina; Bossak, Karolina; Stefaniak, Ewelina; Hureau, Christelle; Raibaut, Laurent; Bal, Wojciech; Faller, Peter
    Chemistry - A European Journal (2018), 24 (32), 8029-8041CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Peptides and proteins with N-terminal amino acid sequences NH2-Xxx-His (XH) and NH2-Xxx-Zzz-His (XZH) form well-established high-affinity CuII-complexes. Key examples are Asp-Ala-His (in serum albumin) and Gly-His-Lys, the wound-healing factor. This opens a straightforward way to add a high-affinity CuII-binding site to almost any peptide or protein, by chem. or recombinant approaches. Thus, these motifs, NH2-Xxx-Zzz-His in particular, have been used to equip peptides and proteins with a multitude of functions based on the redox activity of Cu, including nuclease, protease, glycosidase, or oxygen activation properties, useful in anticancer or antimicrobial drugs. More recent research suggests novel biol. functions, mainly based on the redox inertness of CuII in XZH, like PET imaging (with 64Cu), chelation therapies (for instance in Alzheimer's disease and other types of neurodegeneration), antioxidant units, Cu transporters, and activation of biol. functions by strong CuII binding. Here, the authors provide an overview of the chem. properties of Cu-XH and -XZH motifs and discuss the pros and cons of the vastly different biol. applications, and how they could be improved depending on the application.
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  19. 19
    Nagaj, J.; Stokowa-Sołtys, K.; Zawisza, I.; Jeżowska-Bojczuk, M.; Bonna, A.; Bal, W. Selective control of Cu(II) complex stability in histidine peptides by β-alanine. J. Inorg. Biochem. 2013, 119, 85– 89,  DOI: 10.1016/j.jinorgbio.2012.11.002
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    Selective control of Cu(II) complex stability in histidine peptides by β-alanine
    Nagaj, Justyna; Stokowa-Soltys, Kamila; Zawisza, Izabela; Jezowska-Bojczuk, Malgorzata; Bonna, Arkadiusz; Bal, Wojciech
    Journal of Inorganic Biochemistry (2013), 119 (), 85-89CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)
    The cooperativity of formation of 5-membered and 6-membered chelate rings is the driving force for specificity and selectivity in Cu(II) peptidic complexes. α-Amino acids enable the formation of 5-membered rings, while a 6-membered ring is provided by the coordination of the His side chain imidazole. Introduction of β-alanine is another way of creating a 6-membered ring in the Cu(II) complex. The potentiometric and spectroscopic (UV-vis and CD) study of Cu(II) complexation by a series of four peptides, AAH-am, ABH-am, BAH-am, and BBH-am (where B stands for β-alanine, and -am for C-terminal amide) revealed a very strong effect of the sizes of individual rings, with the order of complex stability AAH-am (5,5,6) > BAH-am (6,5,6) > ABH-am (5,6,6) » BBH-am (6,6,6). The stabilities of ABH-am and BAH-am complexes are intermediate between those of strong His-3 peptides but these complexes are still able to sat. the coordination sphere of the Cu(II) ion at neutral pH. This fact opens up new possibilities in engineering specific peptide-based chelates.
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  20. 20
    Kotuniak, R.; Strampraad, M. J. F.; Bossak-Ahmad, K.; Wawrzyniak, U. E.; Ufnalska, I.; Hagedoorn, P.-L.; Bal, W. Key Intermediate Species Reveal the Copper(II)-Exchange Pathway in Biorelevant ATCUN/NTS Complexes. Angew. Chem., Int. Ed. 2020, 59, 11234– 11239,  DOI: 10.1002/anie.202004264
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    20
    Key Intermediate Species Reveal the Copper(II)-Exchange Pathway in Biorelevant ATCUN/NTS Complexes
    Kotuniak, Radoslaw; Strampraad, Marc J. F.; Bossak-Ahmad, Karolina; Wawrzyniak, Urszula E.; Ufnalska, Iwona; Hagedoorn, Peter-Leon; Bal, Wojciech
    Angewandte Chemie, International Edition (2020), 59 (28), 11234-11239CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The amino-terminal copper and nickel/N-terminal site (ATCUN/NTS) present in proteins and bioactive peptides exhibits high affinity towards CuII ions and have been implicated in human copper physiol. Little is known, however, about the rate and exact mechanism of formation of such complexes. We used the stopped-flow and microsecond freeze-hyperquenching (MHQ) techniques supported by steady-state spectroscopic and electrochem. data to demonstrate the formation of partially coordinated intermediate CuII complexes formed by glycyl-glycyl-histidine (GGH) peptide, the simplest ATCUN/NTS model. One of these novel intermediates, characterized by two-nitrogen coordination, t1/2≈100 ms at pH 6.0 and the ability to maintain the CuII/CuI redox pair is the best candidate for the long-sought reactive species in extracellular copper transport.
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    Bossak-Ahmad, K.; Frączyk, T.; Bal, W.; Drew, S. C. The Sub-picomolar Cu2+ Dissociation Constant of Human Serum Albumin. ChemBioChem 2020, 21, 331– 334,  DOI: 10.1002/cbic.201900435
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    21
    The Sub-picomolar Cu2+ Dissociation Constant of Human Serum Albumin
    Bossak-Ahmad, Karolina; Fraczyk, Tomasz; Bal, Wojciech; Drew, Simon C.
    ChemBioChem (2020), 21 (3), 331-334CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The apparent affinity of human serum albumin (HSA) for divalent copper has long been the subject of great interest, due to its presumed role as the major Cu2+-binding ligand in blood and cerebrospinal fluid. Using a combination of electronic absorption, CD and room-temp. ESR spectroscopies, together with potentiometric titrns., we competed the tripeptide GGH against HSA to reveal a conditional binding const. of log cKCuCu(HSA)=13.02±0.05 at pH 7.4. This rigorously detd. value of the Cu2+ affinity has important implications for understanding the extracellular distribution of copper.
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    Magrì, A.; Tabbì, G.; Giuffrida, A.; Pappalardo, G.; Satriano, C.; Naletova, I.; Nicoletti, V. G.; Attanasio, F. Influence of the N-terminus acetylation of Semax, a synthetic analog of ACTH(4–10), on copper(II) and zinc(II) coordination and biological properties. J. Inorg. Biochem. 2016, 164, 59– 69,  DOI: 10.1016/j.jinorgbio.2016.08.013
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    22
    Influence of the N-terminus acetylation of Semax, a synthetic analog of ACTH(4-10), on copper(II) and zinc(II) coordination and biological properties
    Magri, Antonio; Tabbi, Giovanni; Giuffrida, Alessandro; Pappalardo, Giuseppe; Satriano, Cristina; Naletova, Irina; Nicoletti, Vincenzo G.; Attanasio, Francesco
    Journal of Inorganic Biochemistry (2016), 164 (), 59-69CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)
    Semax is a heptapeptide (Met-Glu-His-Phe-Pro-Gly-Pro) that encompasses the sequence 4-7 of N-terminal domain of the adrenocorticotropic hormone and a C-terminal Pro-Gly-Pro tripeptide. N-terminal amino group acetylation (Ac-Semax) modulates the chem. and biol. properties of parental peptide, modifying the ability of Semax to form complex species with Cu(II) ion. At physiol. pH, the main complex species formed by Ac-Semax, [CuLH-2]2-, consists in a distorted CuN3O chromophore with a weak apical interaction of the methionine sulfur. Such a complex differs from the Cu(II)-Semax complex system, which exhibits a CuN4 chromophore. The reduced ligand field affects the [CuLH-2]2- formal redox potential, which is more pos. than that of Cu(II)-Semax corresponding species. In the amino-free form, the resulting complex species is redox-stable and unreactive against ascorbic acid, unlike the acetylated form. Semax acetylation did not protect from Cu(II) induced toxicity on a SH-SY5Y neuroblastoma cell line, thus demonstrating the crucial role played by the free NH2 terminus in the cell protection. Since several brain diseases are assocd. either to Cu(II) or Zn(II) dyshomeostasis, here we characterized also the complex species formed by Zn(II) with Semax and Ac-Semax. Both peptides were able to form Zn(II) complex species with comparable strength. Confocal microscopy imaging confirmed that peptide group acetylation does not affect the Zn(II) influx in neuroblastoma cells. Moreover, a punctuate distribution of Zn(II) within the cells suggests a preferred subcellular localization that might explain the zinc toxic effect. A future perspective can be the use of Ac-Semax as ionophore in antibody drug conjugates to produce a dysmetallostasis in tumor cells.
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    Gavriilidou, A. F. M.; Gülbakan, B.; Zenobi, R. Influence of Ammonium Acetate Concentration on Receptor-Ligand Binding Affinities Measured by Native Nano ESI-MS: A Systematic Study. Anal. Chem. 2015, 87, 10378– 10384,  DOI: 10.1021/acs.analchem.5b02478
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    Influence of Ammonium Acetate Concentration on Receptor-Ligand Binding Affinities Measured by Native Nano ESI-MS: A Systematic Study
    Gavriilidou, Agni F. M.; Gulbakan, Basri; Zenobi, Renato
    Analytical Chemistry (Washington, DC, United States) (2015), 87 (20), 10378-10384CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Native electrospray ionization (ESI) mass spectrometry (MS) is a powerful technique for analyzing biomols. in their native state. However, ESI-MS is incompatible with nonvolatile soln. additives. Therefore, biomols. have to be electrosprayed from a soln. that differs from their purifn. or storage buffer, often aq. ammonium acetate (AmAc). In this study, the effect of the ionic strength on the dissocn. consts. of six different noncovalent complexes, that cover interactions present in many biol. systems, was investigated. Complexes were electrosprayed from 10 mM, 50 mM, 100 mM, 300 mM, and 500 mM aq. AmAc. For all systems, it was shown that the binding affinity is significantly influenced by the ionic strength of the soln. The detd. dissocn. const. (Kd) was affected more than 50% when increasing the AmAc concn. The results are interpreted in terms of altered ionic interactions induced by the soln. This work emphasizes the modulating effect of the ions on noncovalent interactions and the importance of carefully choosing the AmAc concn. for quantifying the receptor-ligand binding strengths.
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    Erba, E. B.; Zenobi, R. Mass spectrometric studies of dissociation constants of noncovalent complexes. Annu. Rep. Prog. Chem., Sect. C: Phys. Chem. 2011, 107, 199,  DOI: 10.1039/c1pc90006d
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    Mass spectrometric studies of dissociation constants of noncovalent complexes
    Erba, Elisabetta Boeri; Zenobi, Renato
    Annual Reports on the Progress of Chemistry, Section C: Physical Chemistry (2011), 107 (), 199-228CODEN: ACPCDW; ISSN:0260-1826. (Royal Society of Chemistry)
    A review. Specific interactions among biomols. to form noncovalently bound complexes play a pivotal role in key cellular processes such as cell division, cell signalling, gene transcription and translation. The propensity of noncovalently bound complexes to dissoc. into their components can be quantified and consts. of dissocn. (Kd) can be obtained by various methods. Mass spectrometry has become an important method to measure Kd. The advent of soft ionisation techniques, in particular electrospray ionisation (ESI) and matrix-assisted laser desorption/ionisation (MALDI) has established mass spectrometry as a viable technique for investigating noncovalent interactions and for quantifying their binding strengths. Under carefully chosen exptl. and instrumental conditions, it is possible to observe intact noncovalent complexes in the gas phase using ESI and MALDI, and to use the mass spectra as a read-out for detg. soln.-phase Kd. Compared to other biophys. methods, mass spectrometry is highly sensitive and fast, and gives addnl. information about the stoichiometry and specificity of noncovalent interactions. This review focuses on recent MS-based methodologies for quantification of binding strengths, in particular those that promise to complement conventional biophys. methods.
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    Daniel, J. M.; McCombie, G.; Wendt, S.; Zenobi, R. Mass spectrometric determination of association constants of adenylate kinase with two noncovalent inhibitors. J. Am. Soc. Mass Spectrom. 2003, 14, 442– 448,  DOI: 10.1016/S1044-0305(03)00132-6
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    Mass spectrometric determination of association constants of adenylate kinase with two noncovalent inhibitors
    Daniel, J. urg M.; McCombie, Gregor; Wendt, Silke; Zenobi, Renato
    Journal of the American Society for Mass Spectrometry (2003), 14 (5), 442-448CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Science Inc.)
    Noncovalent complexes between chicken muscle adenylate kinase and two inhibitors, P1,P4-di(adenosine-5')tetraphosphate (Ap4A) and P1,P5-di(adenosine-5') pentaphosphate (Ap5A), were investigated with electrospray ionization mass spectrometry under non-denaturing conditions. The noncovalent nature and the specificity of the complexes are demonstrated with a no. of control expts. Titrn. expts. allowed the assocn. consts. for inhibitor binding to be detd. Problems with concn. dependent ion yields are circumvented by a data evaluation method that is insensitive to the overall ionization efficiency. The Ka values found were 9.0 × 104 M-1 (Ap4A) and 4.0 × 107 M-1 (Ap5A), resp., in very good agreement with available literature data.
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    Peschke, M.; Verkerk, U. H.; Kebarle, P. Features of the ESI mechanism that affect the observation of multiply charged noncovalent protein complexes and the determination of the association constant by the titration method. J. Am. Soc. Mass Spectrom. 2004, 15, 1424– 1434,  DOI: 10.1016/j.jasms.2004.05.005
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    26
    Features of the ESI mechanism that affect the observation of multiply charged noncovalent protein complexes and the determination of the association constant by the titration method
    Peschke, Michael; Verkerk, Udo H.; Kebarle, Paul
    Journal of the American Society for Mass Spectrometry (2004), 15 (10), 1424-1434CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)
    Several factors, attributable to the ESIMS mechanism, that can affect the assumptions of the titrn. method are examd.: (1) The assumption that the concns. in soln. of the protein P, the ligand L, and the complex PL are proportional to the resp. ion intensities obsd. with ESIMS, is examd. with expts. in which ion intensities of two non-interacting proteins are compared with the resp. concns. The intensities are found to be approx. proportional to the concns. The proportionality factors are found to increase as the mass of the protein is decreased. Very small proteins have much higher intensities. The results suggest that it is preferable to use only the intensity ratio of PL and P, whose masses are very close to each other when L is small, to det. the assocn. const. KA in soln. (2) From the charge residue model (CRM) one expects that the soln. will experience a very large increase of concn. due to evapn. of the precursor droplets, before the proteins P and PL are produced in the gas phase. This can shift the equil. in the droplets: P + L = PL, towards PL. Anal. of the droplet evapn. history shows that such a shift is not likely, because the time of droplet evolution is very short, only several μs, and the equil. relaxation time is much longer. (3) The droplet history shows that unreacted P and L can be often present together in the same droplet. On complete evapn. of such droplets L will land on P leading to PL and this effect will lead to values of KA that are too high. However, it is argued that mostly accidental, weakly bonded, complexes will form and these will dissoc. in the clean up stages (heated transfer capillary and CAD region). Thus only very small errors are expected due to this cause. (4) Some PL complexes may have bonding that is too weak in the gas phase even though they have KA values in soln. that predict high soln. PL yields. In this case the PL complexes may decomp. in the clean up stages and not be obsd. with sufficient intensity in the mass spectrum. This will lead to KA values that are too low. The effect is expected for complexes that involve significant hydrophobic interaction that leads to high stability of the complex in soln. but low stability in the gas phase. The titrn. method is not suited for such systems.
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  27. 27
    Zhang, S.; van Pelt, C. K.; Wilson, D. B. Quantitative determination of noncovalent binding interactions using automated nanoelectrospray mass spectrometry. Anal. Chem. 2003, 75, 3010– 3018,  DOI: 10.1021/ac034089d
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    Quantitative determination of noncovalent binding interactions using automated nanoelectrospray mass spectrometry
    Zhang, Sheng; Van Pelt, Colleen K.; Wilson, David B.
    Analytical Chemistry (2003), 75 (13), 3010-3018CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Electrospray ionization mass spectrometry (ESI-MS) has proven to be an extremely powerful tool for studying biomol. structures and noncovalent interactions. Here we report a method using a fully automated, chip-based nanoESI-MS system to det. the dissocn. consts. (Kd) for the complexes of two different proteins with their ligands. The automated nanoelectrospray system, consisting of the NanoMate and ESI chip, serves functionally as a combination of autosampler and nanoelectrospray ionization source. This system provides all the advantages of conventional nanoelectrospray plus automated, high-throughput analyses without carryover. The automated nanoESI system was used to investigate quant. noncovalent interactions between RNase A (RNase A) and cytidylic acid ligands (2'-CMP, CTP), a well-characterized model protein-ligand complex, and between an inactive endocellulase mutant (Thermobifida fusca Cel6A D117Acd) and four oligosaccharide ligands (cellotriose, cellotetraose, cellopentaose, cellohexaose). Both titrn. and competitive binding approaches were performed prior to automated nanoESI-MS anal. with a Q-TOF mass spectrometer. Dissocn. consts. for each complex were calcd. from the sum of ion peak areas of free and complexed proteins during the titrn. and competition expts. The measured Kd values for the RNase A-CMP and Cel6A D117Acd-G3 complexes were found to be in excellent agreement with the available published values obtained by std. spectroscopic titrn. techniques. To our knowledge, this is the first report of using an ESI-MS approach to study the interactions between a cellulase and oligosaccharides. The results provide new insights for understanding the nature of cellulase-cellulose interactions.
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  28. 28
    Carlton, D. D., Jr; Schug, K. A. A review on the interrogation of peptide-metal interactions using electrospray ionization-mass spectrometry. Anal. Chim. Acta 2011, 686, 1– 39,  DOI: 10.1016/j.aca.2010.11.050
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    Jecklin, M. C.; Touboul, D.; Bovet, C.; Wortmann, A.; Zenobi, R. Which electrospray-based ionization method best reflects protein-ligand interactions found in solution? a comparison of ESI, nanoESI, and ESSI for the determination of dissociation constants with mass spectrometry. J. Am. Soc. Mass Spectrom. 2008, 19, 332– 343,  DOI: 10.1016/j.jasms.2007.11.007
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    29
    Which electrospray-based ionization method best reflects protein-ligand interactions found in solution? A comparison of ESI, nanoESI, and ESSI for the determination of dissociation constants with mass spectrometry
    Jecklin, Matthias Conradin; Touboul, David; Bovet, Cedric; Wortmann, Arno; Zenobi, Renato
    Journal of the American Society for Mass Spectrometry (2008), 19 (3), 332-343CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)
    The authors present a comparison of three different electrospray-based ionization techniques for the investigation of noncovalent complexes with mass spectrometry. The features and characteristics of std. electrospray ionization (ESI), chip-based nanoESI, and electrosonic spray ionization (ESSI) mounted onto a hybrid quadrupole time-of-flight mass spectrometer were compared in their performance to det. the dissocn. const. (KD) of the model system hen egg white lysozyme (HEWL) binding to N,N',N''-triacetylchitotriose (NAG3). The best KD value compared with soln. data were found for ESSI, 19.4 ± 3.6 μM. Then, the authors detd. the KDs of the two nucleotide binding sites of adenylate kinase (AK), where the authors obtained KDs of 2.2 ± 0.8 μM for the first and 19.5 ± 8.0 μM for the second binding site using ESSI. The authors found a weak charge state dependence of the KD for both protein-ligand systems, where for all ionization techniques the KD value decreases with increasing charge state. The authors demonstrate that ESSI is very gentle and insensitive to instrumental parameters, and the KD obtained is in good agreement with soln. phase results from the literature. In addn., the authors tried to det. the KD for the lymphocyte-specific kinase LCK binding to a kinase inhibitor using nanoESI due to the very low amt. of sample available. In this case, the authors found KD values with a strong charge state dependence, which were in no case close to literature values for soln. phase.
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  30. 30
    Kitova, E. N.; El-Hawiet, A.; Schnier, P. D.; Klassen, J. S. Reliable Determinations of Protein-Ligand Interactions by Direct ESI-MS Measurements. Are We There Yet?. J. Am. Soc. Mass Spectrom. 2012, 23, 431– 441,  DOI: 10.1007/s13361-011-0311-9
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    30
    Reliable determinations of protein-ligand interactions by direct ESI-MS measurements. Are we there yet?
    Kitova, Elena N.; El-Hawiet, Amr; Schnier, Paul D.; Klassen, John S.
    Journal of the American Society for Mass Spectrometry (2012), 23 (3), 431-441CODEN: JAMSEF; ISSN:1044-0305. (Springer)
    A review. The assocn.-dissocn. of noncovalent interactions between protein and ligands, such as other proteins, carbohydrates, lipids, DNA, or small mols., are crit. events in many biol. processes. The discovery and characterization of these interactions is essential to a complete understanding of biochem. reactions and pathways and to the design of novel therapeutic agents that may be used to treat a variety of diseases and infections. Over the last 20 y, electrospray ionization mass spectrometry (ESI-MS) has emerged as a versatile tool for the identification and quantification of protein-ligand interactions in vitro. Here, the authors describe the implementation of the direct ESI-MS assay for the detn. of protein-ligand binding stoichiometry and affinity. Addnl., the authors outline common sources of error encountered with these measurements and various strategies to overcome them. Finally, the authors comment on some of the outstanding challenges assocd. with the implementation of the assay and highlight new areas where direct ESI-MS measurements are expected to make significant contributions in the future.
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  31. 31
    Smirnova, J.; Zhukova, L.; Witkiewicz-Kucharczyk, A.; Kopera, E.; Olędzki, J.; Wysłouch-Cieszyńska, A.; Palumaa, P.; Hartwig, A.; Bal, W. Quantitative electrospray ionization mass spectrometry of zinc finger oxidation: The reaction of XPA zinc finger with H2O2. Anal. Biochem. 2007, 369, 226– 231,  DOI: 10.1016/j.ab.2007.05.019
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    Quantitative electrospray ionization mass spectrometry of zinc finger oxidation: The reaction of XPA zinc finger with H2O2
    Smirnova, Julia; Zhukova, Liliya; Witkiewicz-Kucharczyk, Aleksandra; Kopera, Edyta; Oledzki, Jacek; Wyslouch-Cieszynska, Aleksandra; Palumaa, Peep; Hartwig, Andrea; Bal, Wojciech
    Analytical Biochemistry (2007), 369 (2), 226-231CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)
    Oxidn. plays an important role in the functioning of zinc fingers (ZFs). Electrospray ionization mass spectrometry (ESI-MS) is a very useful technique to study products of ZF oxidn., but its application has been limited largely to qual. anal. of reaction products. ESI-MS has been applied successfully on several occasions to det. binding consts. in metalloproteins. The authors used a synthetic 37-residue peptide acetyl-DYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf), which corresponds to the Cys 4 ZF sequence of human nucleotide excision repair protein XPA, to find out whether ESI-MS might be used quant. to study ZF reaction kinetics. For this purpose, the authors studied oxidn. of the Zn(II) complex of XPAzf (ZnXPAzf) by H2O2 using three techniques in parallel: HPLC of covalent reaction products, 4-(2-pyridylazo)-resorcinol monosodium salt (PAR)-based spectrophotometric zinc release assay, and ESI-MS. Single and double intrapeptide disulfides were detected by ESI-MS to be the sole reaction products. All three techniques yielded independently the same reaction rate, thereby demonstrating that ESI-MS may indeed be used in quant. kinetic studies of ZF reactions. The comparison of exptl. information demonstrated that the formation of the Cys 5Cys 8 single disulfide was responsible for zinc release.
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    Shoshan, M. S.; Dekel, N.; Goch, W.; Shalev, D. E.; Danieli, T.; Lebendiker, M.; Bal, W.; Tshuva, E. Y. Unbound position II in MXCXXC metallochaperone model peptides impacts metal binding mode and reactivity: Distinct similarities to whole proteins. J. Inorg. Biochem. 2016, 159, 29– 36,  DOI: 10.1016/j.jinorgbio.2016.02.016
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    Unbound position II in MXCXXC metallochaperone model peptides impacts metal binding mode and reactivity: Distinct similarities to whole proteins
    Shoshan, Michal S.; Dekel, Noa; Goch, Wojciech; Shalev, Deborah E.; Danieli, Tsafi; Lebendiker, Mario; Bal, Wojciech; Tshuva, Edit Y.
    Journal of Inorganic Biochemistry (2016), 159 (), 29-36CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)
    The effect of position II in the binding sequence of copper metallochaperones, which varies between Thr and His, was investigated through structural anal. and affinity and oxidn. kinetic studies of model peptides. A first Cys-Cu(I)-Cys model obtained for the His peptide at acidic and neutral pH, correlated with higher affinity and more rapid oxidn. of its complex; in contrast, the Thr peptide with the Cys-Cu(I)-Met coordination under neutral conditions demonstrated weaker and pH dependent binding. Studies with human antioxidant protein 1 (Atox1) and three of its mutants where S residues were replaced with Ala suggested that (a) the binding affinity is influenced more by the binding sequence than by the protein fold (b) pH may play a role in binding reactivity, and (c) mutating the Met impacted the affinity and oxidn. rate more drastically than did mutating one of the Cys, supporting its important role in protein function. Position II thus plays a dominant role in metal binding and transport.
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    Piątek, K.; Schwerdtle, T.; Hartwig, A.; Bal, W. Monomethylarsonous Acid Destroys a Tetrathiolate Zinc Finger Much More Efficiently than Inorganic Arsenite: Mechanistic Considerations and Consequences for DNA Repair Inhibition. Chem. Res. Toxicol. 2008, 21, 600– 606,  DOI: 10.1021/tx7003135
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    Monomethylarsonous Acid Destroys a Tetrathiolate Zinc Finger Much More Efficiently than Inorganic Arsenite: Mechanistic Considerations and Consequences for DNA Repair Inhibition
    Piatek, Katarzyna; Schwerdtle, Tanja; Hartwig, Andrea; Bal, Wojciech
    Chemical Research in Toxicology (2008), 21 (3), 600-606CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
    Arsenic compds. are human carcinogens. The ingested inorg. arsenic is metabolized to methylated derivs., which are considered to be more toxic than the inorg. species. Interactions of trivalent arsenicals with thiol groups of proteins are believed to be important for arsenic carcinogenesis, but inorg. arsenite appears to bind to thiol groups more strongly than the methylated AsIII species. Inhibition of the nucleotide excision repair pathway of DNA repair (NER) is likely to be of primary importance in arsenic carcinogenesis. Previously, we demonstrated that methylated AsIII compds. are more efficient than arsenite in releasing zinc from ZnXPAzf, the zinc finger of XPA, a crucial member of the NER complex. In this work, we used ESI-MS to compare aerobic reactivities of arsenite and monomethylarsonous acid (MMAIII) toward ZnXPAzf on the mol. level. We demonstrated that equimolar MMAIII released ZnII from ZnXPAzf easily, forming mono- and diarsenical derivs. of XPAzf. This reaction was accompanied by oxidn. of unprotected thiol groups of the monomethylarsinated peptide to intramol. disulfides. The estd. affinity of MMAIII to XPAzf is 30-fold higher than that established previously for arsenite binding to the thiol groups. No binding of arsenite to the thiol groups of XPAzf was obsd. under our exptl. conditions, and a 10-fold excess of arsenite was required to partially oxidize ZnXPAzf. These results indicate a particular susceptibility of tetrathiolate zinc fingers to MMAIII, thereby providing a novel mol. pathway in arsenic carcinogenesis.
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    Whittal, R. M.; Ball, H. L.; Cohen, F. E.; Burlingame, A. L.; Prusiner, S. B.; Baldwin, M. A. Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry. Protein Sci. 2000, 9, 332– 343,  DOI: 10.1110/ps.9.2.332
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    Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry
    Whittal, Randy M.; Ball, Haydn L.; Cohen, Fred E.; Burlingame, Alma L.; Prusiner, Stanley B.; Baldwin, Michael A.
    Protein Science (2000), 9 (2), 332-343CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)
    Electrospray ionization mass spectrometry (ESI-MS) was used to measure the binding of Cu2+ ions to synthetic peptides corresponding to sections of the sequence of the mature prion protein (PrP). ESI-MS demonstrates that Cu2- is unique among divalent metal ions in binding to PrP and defines the location of the major Cu2+ binding site as the octarepeat region in the N-terminal domain, contg. multiple copies of the repeat ProHisGlyGlyGlyTrpGlyGln. The stoichiometries of the complexes measured directly by ESI-MS are pH dependent: a peptide contg. four octarepeats chelates two Cu2+ ions at pH 6 but four at pH 7.4. At the higher pH, the binding of multiple Cu2+ ions occurs with a high degree of cooperativity for peptides C-terminally extended to incorporate a fifth histidine. Dissocn. consts. for each Cu2+ ion binding to the octarepeat peptides, reported here for the first time, are mostly in the low micromolar range; for the addn. of the third and fourth Cu2+ ions to the extended peptides at pH 7.4, KDs are <100 nM. N-terminal acetylation of the peptides caused some redn. in the stoichiometry of binding at both pHs. Cu2+ also binds to a peptide corresponding to the extreme N-terminus of PrP that precedes the octarepeats, arguing that this region of the sequence may also make a contribution to the Cu2+ complexation. Although the structure of the four-octarepeat peptide is not affected by pH changes in the absence of Cu2+, as judged by CD, Cu2+ binding induces a modest change at pH 6 and a major structural perturbation at pH 7.4. It is possible that PrP functions as a Cu2+ transporter by binding Cu2+ ions from the extracellular medium under physiol. conditions and then releasing some or all of this metal upon exposure to acidic pH in endosomes or secondary lysosomes.
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    Di Marco, V. B.; Bombi, G. G. Electrospray mass spectrometry (ESI-MS) in the study of metal-ligand solution equilibria. Mass Spectrom. Rev. 2006, 25, 347– 379,  DOI: 10.1002/mas.20070
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    Electrospray mass spectrometry (ESI-MS) in the study of metal-ligand solution equilibria
    Di Marco, Valerio B.; Bombi, G. Giorgio
    Mass Spectrometry Reviews (2006), 25 (3), 347-379CODEN: MSRVD3; ISSN:0277-7037. (John Wiley & Sons, Inc.)
    A review. In the 20 years, since the introduction of electrospray mass spectrometry (ESI-MS), the use of this technique in various fields of inorg., organometallic, and anal. chem. was steadily increasing. The application of ESI-MS to the study of metal-ligand soln. equil. is reviewed (till 2004 included). In a 1st section, advantages and drawbacks of ESI-MS in this type of application are described. Subsequently, a list of ∼300 studies is reported, in which ESI-MS was used to give no. and stoichiometry of the species at equil., or also to est. their stability consts. All studies are classified according to the metal ions under examn. Other related applications, such as host-guest interactions and metal ion-protein binding studies, are briefly reviewed as well.
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    Wyttenbach, T.; Liu, D.; Bowers, M. T. Interactions of the hormone oxytocin with divalent metal ions. J. Am. Chem. Soc. 2008, 130, 5993– 6000,  DOI: 10.1021/ja8002342
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    Interactions of the Hormone Oxytocin with Divalent Metal Ions
    Wyttenbach, Thomas; Liu, Dengfeng; Bowers, Michael T.
    Journal of the American Chemical Society (2008), 130 (18), 5993-6000CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The interaction of the cyclic nonapeptide oxytocin (OT) with a no. of alk. earth and divalent transition metal ions (X2+) was examd. employing mass spectrometry (MS) and ion mobility spectrometry (IMS) techniques in combination with mol. dynamics (MD) and d. functional theory (DFT) calcns. Under acidic conditions OT exhibits an exceptionally strong affinity for all divalent metal ions resulting in strong [OT + X]2+ peaks in the mass spectrum. Under basic conditions only Cu2+ and Ni2+-OT complexes were detected and these were singly, doubly, triply, or quadruply deprotonated. Collision-induced dissocn. of the [OT - 3H + Cu]- complex yielded exclusively C-terminal Cu2+-contg. fragments (Cu2+fragment3-), suggesting that the Cu2+ ligation site includes deprotonated C-terminal backbone amide nitrogen atoms and the N-terminal amino nitrogen atom in [OT - 3H + Cu]-. MD and DFT calcns. indicate a square-planar complex is consistent with these observations and with exptl. collision cross sections. MD and DFT calcns. also indicate either an octahedral or trigonal-bipyramidal complex between Zn2+ and OT is lowest in energy with carbonyl oxygens being the primary ligation sites. Both complexes yield cross sections in agreement with expt. The biol. impact of the structural changes induced in OT by divalent metal ion coordination is discussed.
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    Ikonomou, M. G.; Blades, A. T.; Kebarle, P. Investigations of the Electrospray Interface for Liquid Chromatography/Mass Spectrometry. Anal. Chem. 1990, 62, 957– 967,  DOI: 10.1021/ac00208a012
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    Investigations of the electrospray interface for liquid chromatography/mass spectrometry
    Ikonomou, Michael G.; Blades, Arthur T.; Kebarle, Paul
    Analytical Chemistry (1990), 62 (9), 957-67CODEN: ANCHAM; ISSN:0003-2700.
    The pos. ions, produced by electrospray from solns. of various analytes in methanol, were detected with an atm. pressure triple quadrupole mass spectrometer. Analytes studied included NH4+, Na+, K+, Cs+, Ca2+ and the onium ions BH+ of some 30 org. nitrogen bases B. The analyte ion sensitivities decrease with analyte ion concn. and presence of foreign electrolyte in the soln., at concn. above 10-5 mol/L. At low concns., sensitivities are very high such that ions at 10-8 mol/L concn. can be easily detected. Detection limits in the subfemtomol to attomol range have been achieved. The sensitivity of the org. bases B is pH dependent and increases as the [BH+] in soln. increases. However, the effect is obscured by the depression of the ion signal caused by foreign electrolyte. It is also shown that above 10-5 mol/L B in soln., gaseous B at sufficient pressure can be generated and gas-phase proton transfer to higher gas phase coanalytes can occur. Dimers, B2H+, may also be formed in the gas phase. The gas-phase ion reaction time is estd. as ∼2 ms.
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    Wortmann, A.; Kistler-Momotova, A.; Zenobi, R.; Heine, M. C.; Wilhelm, O.; Pratsinis, S. E. Shrinking droplets in electrospray ionization and their influence on chemical equilibria. J. Am. Soc. Mass Spectrom. 2007, 18, 385– 393,  DOI: 10.1016/j.jasms.2006.10.010
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    Shrinking Droplets in Electrospray Ionization and Their Influence on Chemical Equilibria
    Wortmann, Arno; Kistler-Momotova, Anna; Zenobi, Renato; Heine, Martin C.; Wilhelm, Oliver; Pratsinis, Sotiris E.
    Journal of the American Society for Mass Spectrometry (2007), 18 (3), 385-393CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)
    The authors investigated how chem. equil. are affected by the electrospray process, using simultaneous in situ measurements by laser-induced fluorescence (LIF) and phase Doppler anemometry (PDA). The motivation for this study was the increasing no. of publications in which electrospray ionization mass spectrometry is used for binding const. detn. The PDA was used to monitor droplet size and velocity, whereas LIF was used to monitor fluorescent analytes within the electrospray droplets. Using acetonitrile as solvent, the authors found an av. initial droplet diam. of 10 μm in the electrospray. The PDA allowed the authors to follow the evolution of these droplets down to a size of 1 μm. Rhodamine B-sulfonylchloride was used as a fluorescent analyte within the electrospray. By spatially resolved LIF it was possible to probe the dimerization equil. of this dye. Measurements at different spray positions showed no influence of the decreasing droplet size on the monomer-dimer equil. However, with the fluorescent dye pair DCM and oxazine 1 it was shown that a concn. increase does occur within electrosprayed droplets, using fluorescence resonance energy transfer as a probe for the av. pair distance.
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    Kebarle, P.; Tang, L. From ions in solution to ions in the gas phase-the mechanism of electrospray mass spectrometry. Anal. Chem. 1993, 65, 972A– 986A,  DOI: 10.1021/ac00070a715
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    From ions in solution to ions in the gas phase - the mechanism of electrospray mass spectrometry
    Kebarle, Paul; Tang, Liang
    Analytical Chemistry (1993), 65 (22), 972A-986ACODEN: ANCHAM; ISSN:0003-2700.
    The title topic is reviewed with 44 refs. The subjects include: the electrospray (ES) mechanism, prodn. of charged droplets at the ES capillary tip, shrinkage of charged ES droplets, nature of processes leading to formation of gas-phase ions, details of the Iribarne ion evapn. theory, dependence of ion intensities on concn., effects due to the addn. of 2 electrolytes to the solvent, comparison of coeffs. with Iribarne theory and SIDT (single ion in droplet theory), emission of gas-phase ions from the Taylor tip of the ES capillary, and formation mechanisms of multiply-charged macroions.
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    Wilkins, R. G. Kinetics and mechanism of reactions of transition metal complexes, 2nd ed.; VCH: Weinheim, online resource, 2003.
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    Teng, X.; Stefaniak, E.; Girvan, P.; Kotuniak, R.; Płonka, D.; Bal, W.; Ying, L. Hierarchical binding of copperII to N-truncated Aβ4–16 peptide. Metallomics 2020, 12, 470– 473,  DOI: 10.1039/C9MT00299E
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    Hierarchical binding of copperII to N-truncated Aβ4-16 peptide
    Teng, Xiangyu; Stefaniak, Ewelina; Girvan, Paul; Kotuniak, Radoslaw; Plonka, Dawid; Bal, Wojciech; Ying, Liming
    Metallomics (2020), 12 (4), 470-473CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)
    N-Truncated Aβ4-42 displays a high binding affinity with CuII. A mechanistic scheme of the interactions between Aβ4-42 and CuII has been proposed using a fluorescence approach. The timescales of different conversion steps were detd. This kinetic mechanism indicates the potential synaptic functions of Aβ4-42 during neurotransmission.
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    Matsumoto, A.; Fukumoto, T.; Adachi, H.; Watarai, H. Electrospray ionization mass spectrometry of metal complexes. Gas phase formation of a binuclear copper(II)-5-Br-PADAP complex. Anal. Chim. Acta 1999, 390, 193– 199,  DOI: 10.1016/S0003-2670(99)00222-6
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    Electrospray ionization mass spectrometry of metal complexes. Gas phase formation of a binuclear copper(II)-5-Br-PADAP complex
    Matsumoto, Atsushi; Fukumoto, Takao; Adachi, Hiroshi; Watarai, Hitoshi
    Analytica Chimica Acta (1999), 390 (1-3), 193-200CODEN: ACACAM; ISSN:0003-2670. (Elsevier Science B.V.)
    The complexes formed from Cu(II) and 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (5-Br-PADAP or HL) in aq. MeOH soln. was studied by electrospray ionization mass spectrometry. The soln. of a 1:1 complex of Cu(II) with 5-Br-PADAP showed five peaks assignable to binuclear [Cu2L2(AcO)]+ and mononuclear [CuL]+, [CuL(H2O)]+, [CuL(AcOH)]+ and [CuL(HL)]+ (AcO = acetate). Collision activated dissocn. revealed the relative order of bonding strengths; Cu-L > Cu-HL > CuL-AcOH > CuL-H2O. The peak intensities of the binuclear complex showed 2nd-order dependency on those of the mono complex. As for the soln. of Ni(II)-5-Br-PADAP, no binuclear complex was obsd. in the mass spectra. Thus, [Cu2L2(AcO)]+ was probably formed by the fast gas phase reaction: 2[CuL]++AcO-↹[Cu2L2(AcO)]+.
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    Dominant forces in protein folding
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    Biochemistry (1990), 29 (31), 7133-55CODEN: BICHAW; ISSN:0006-2960.
    A review, with 328 refs., on the nature and magnitudes of dominant forces in protein folding. Contributions to free energy of folding arising from electrostatics, H bonding and van der Waals interactions, intrinsic propensities, and hydrophobic interactions are explored. Also considered are the opposing forces, conformational entropy and electrostatics.
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    Zhou, S.; Cook, K. D. Protonation in electrospray mass spectrometry: Wrong-way-round or right-way-round?. J. Am. Soc. Mass Spectrom. 2000, 11, 961– 966,  DOI: 10.1016/S1044-0305(00)00174-4
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    Protonation in electrospray mass spectrometry: wrong-way-round or right-way-round?
    Zhou, Shaolian; Cook, Kelsey D.
    Journal of the American Society for Mass Spectrometry (2000), 11 (11), 961-966CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Science Inc.)
    The term "wrong-way-round ionization" has been used in studies of electrospray ionization to describe the observation of protonated or deprotonated ions when sampling strongly basic or acidic solns. (resp.) where such ions are not expected to exist in appreciable concns. in soln. Study of the dependence of ionization of the weak base caffeine on the electrospray capillary potential reveals three distinct contributors to wrong-way-round ionization. At near-neutral pH in solns. of low ionic strength, protonation of caffeine results from the surface enrichment of electrolytically produced protons in the surface layer of the droplets from which ions are desorbed. For solns. made strongly basic with ammonia, gas-phase proton transfer from ammonium ions can create protonated caffeine. These two mechanisms have been discussed previously elsewhere. For solns. of high ionic strength at neutral or high pH, the data suggest that discharge-induced ionization is responsible for the prodn. of protonated caffeine. This mechanism probably accounts for some of the wrong-way-round ionization reported elsewhere.
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    Wu, Q.; Gao, J.; Joseph-McCarthy, D.; Sigal, G. B.; Bruce, J. E.; Whitesides, G. M.; Smith, R. D. Carbonic Anhydrase-Inhibitor Binding: From Solution to the Gas Phase. J. Am. Chem. Soc. 1997, 119, 1157– 1158,  DOI: 10.1021/ja9630250
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    Carbonic Anhydrase-Inhibitor Binding: From Solution to the Gas Phase
    Wu, Qinyuan; Gao, Jinming; Joseph-McCarthy, Diane; Sigal, George B.; Bruce, James E.; Whitesides, George M.; Smith, Richard D.
    Journal of the American Chemical Society (1997), 119 (5), 1157-1158CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The binding of para-substituted benzenesulfonamide inhibitors to bovine carbonic anhydrase II (BCAII, EC 4.2.1.1) in the gas phase and in soln. has been studied by electrospray ionization-mass spectrometry and fluorescence spectroscopy, resp. The off-rates of BCAII-inhibitor complexes in soln. are primarily detd. by the hydrophobic interactions between the inhibitor and the enzyme, while their corresponding gas phase stabilities are governed by the polar surface interactions. The results provide insights into the factors governing gas phase stability of the charged complexes, and show that relative stabilities in soln. and the gas phase are substantially different.
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    Wales, T. E.; Engen, J. R. Partial unfolding of diverse SH3 domains on a wide timescale. J. Mol. Biol. 2006, 357, 1592– 1604,  DOI: 10.1016/j.jmb.2006.01.075
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    Partial unfolding of diverse SH3 domains on a wide timescale
    Wales, Thomas E.; Engen, John R.
    Journal of Molecular Biology (2006), 357 (5), 1592-1604CODEN: JMOBAK; ISSN:0022-2836. (Elsevier B.V.)
    SH3 domains are small, modular domains that are found in many proteins, esp. signal transduction proteins such as tyrosine kinases. While much is known about the sequences and tertiary structures of SH3 domains, far less is known about their soln. dynamics. A slow, partial unfolding event that occurs under physiol. conditions was previously identified in the Hck SH3 domain using H-exchange (HX) mass spectrometry (MS). To det. if this unfolding was unique to Hck SH3, HX MS was used to analyze 11 other SH3 domains: 7 SH3 domains from Src-family kinases and 5 SH3 domains from various proteins. A wide variety of unfolding rates were found, with unfolding half-lives ranging from 1 s to 1 h. The Lyn and α-spectrin SH3 domains exhibited slow, partial unfolding in β-strands D and E and part of the RT-loop. Hck SH3 also underwent partial unfolding in the same region, implying that a unique feature in this area of the domains is responsible for the partial unfolding. Partial unfolding was, however, not a function of sequence conservation. Although the Fyn and Yes SH3 domains are very similar to Hck SH3 in sequence, they exhibited no evidence of partial unfolding. Overall, the results suggest that while the tertiary structure of SH3 domains is highly conserved, the dynamics of SH3 domains are variable.
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    Orte, A.; Craggs, T. D.; White, S. S.; Jackson, S. E.; Klenerman, D. Evidence of an intermediate and parallel pathways in protein unfolding from single-molecule fluorescence. J. Am. Chem. Soc. 2008, 130, 7898– 7907,  DOI: 10.1021/ja709973m
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    Evidence of an Intermediate and Parallel Pathways in Protein Unfolding from Single-Molecule Fluorescence
    Orte, Angel; Craggs, Timothy D.; White, Samuel S.; Jackson, Sophie E.; Klenerman, David
    Journal of the American Chemical Society (2008), 130 (25), 7898-7907CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Detg. how proteins fold into their native structures is a subject of great importance, since ultimately it will allow protein structure and function to be predicted from primary sequence data. In addn., there is now a clear link between protein unfolding and misfolding events and many disease states. However, since proteins fold over rugged, multidimensional energy landscapes, this is a challenging exptl. and theor. problem. Single-mol. fluorescence methods developed over the past decade have the potential to follow the unfolding/folding of individual mols. Mapping out the landscape without ensemble averaging will enable the identification of intermediate states which may not be significantly populated, in addn. to the presence of multiple pathways. To date, there have been only a limited no. of single-mol. folding/unfolding studies under nonequil. conditions and no intermediates have been obsd. Here, for the first time, we present a single-mol. study of the unfolding of a large autofluorescent protein, Citrine, a variant of green fluorescent protein. Single-mol. fluorescence techniques are used to directly detect an intermediate on the unfolding/folding pathway and the existence of parallel unfolding pathways. This work, and the novel methods used, shows that single-mol. fluorescence can now provide new, hitherto exptl. inaccessible, insights into the folding/unfolding of proteins.
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    Kubelka, J.; Eaton, W. A.; Hofrichter, J. Experimental Tests of Villin Subdomain Folding Simulations. J. Mol. Biol. 2003, 329, 625– 630,  DOI: 10.1016/S0022-2836(03)00519-9
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    Experimental Tests of Villin Subdomain Folding Simulations
    Kubelka, Jan; Eaton, William A.; Hofrichter, James
    Journal of Molecular Biology (2003), 329 (4), 625-630CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Science Ltd.)
    We have used laser temp.-jump to investigate the kinetics and mechanism of folding the 35 residue subdomain of the villin headpiece. The relaxation kinetics are biphasic with a sub-microsecond phase corresponding to a helix-coil transition and a slower microsecond phase corresponding to overall unfolding/refolding. At 300 K, the folding time is 4.3(±0.6) μs, making it the fastest folding, naturally occurring protein, with a rate close to the theor. speed limit. This time is in remarkable agreement with the prediction of 5 (+11,-3) μs by Zagrovic et al. from atomistic mol. dynamics simulations using an implicit solvent model. We test their prediction that replacement of the C-terminal phenylalanine residue with alanine will increase the folding rate by removing a transient non-native interaction. We find that the alanine substitution has no effect on the folding rate or on the equil. const. Implications of this result for the validity of the simulated folding mechanism are discussed.
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    Trapaidze, A.; Hureau, C.; Bal, W.; Winterhalter, M.; Faller, P. Thermodynamic study of Cu2+ binding to the DAHK and GHK peptides by isothermal titration calorimetry (ITC) with the weaker competitor glycine. JBIC, J. Biol. Inorg. Chem. 2012, 17, 37– 47,  DOI: 10.1007/s00775-011-0824-5
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    Thermodynamic study of Cu2+ binding to the DAHK and GHK peptides by isothermal titration calorimetry (ITC) with the weaker competitor glycine
    Trapaidze, Ana; Hureau, Christelle; Bal, Wojciech; Winterhalter, Mathias; Faller, Peter
    JBIC, Journal of Biological Inorganic Chemistry (2012), 17 (1), 37-47CODEN: JJBCFA; ISSN:0949-8257. (Springer)
    The peptides Asp-Ala-His-Lys (DAHK) and Gly-His-Lys (GHK) are naturally occurring Cu(II)-chelating motifs in human serum and cerebrospinal fluid. Here, the sensitive thermodn. technique isothermal titrn. calorimetry was used to study the energetics of Cu(II) binding to DAHK and GHK peptides in the presence of the weaker ligand glycine as a competitor. DAHK and GHK bind Cu(II) predominantly in a 1:1 stoichiometry with conditional dissocn. consts. [i.e., at pH 7.4, in the absence of the competing chelators glycine and 2-(4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid buffer] of 2.6 ± 0.4 × 10-14 M and 7.0 ± 1.0 × 10-14 M, resp. Furthermore, the apparent ΔH values were measured and the no. of protons released upon Cu(II) binding was detd. by performing expts. in different buffers. This allowed us to det. the conditional ΔG, ΔH, and ΔS, i.e., cor. for the contributions of the weaker ligand glycine and the buffer at pH 7.4. We found that the entropic and enthalpic contributions to the Cu(II) binding to GHK and DAHK are distinct, with a enthalpic contribution for GHK. The thermodn. parameters obtained correspond well to those in the literature obtained by other techniques, suggesting that the use of the weaker ligand glycine as a competitor in isothermal titrn. calorimetry provides accurate data for Cu(II) binding to high-affinity peptides, which cannot be accurately detd. without the use of a competitor ligand.
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    North, M. L.; Wilcox, D. E. Shift from Entropic Cu2+ Binding to Enthalpic Cu+ Binding Determines the Reduction Thermodynamics of Blue Copper Proteins. J. Am. Chem. Soc. 2019, 141, 14329– 14339,  DOI: 10.1021/jacs.9b06836
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    Shift from Entropic Cu2+ Binding to Enthalpic Cu+ Binding Determines the Reduction Thermodynamics of Blue Copper Proteins
    North, Molly L.; Wilcox, Dean E.
    Journal of the American Chemical Society (2019), 141 (36), 14329-14339CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The enthalpic and entropic components of Cu2+ and Cu+ binding to the blue copper protein azurin have been quantified with isothermal titrn. calorimetry (ITC) measurements and anal., providing the first such exptl. values for Cu+ binding to a protein. The high affinity of azurin for Cu2+ is entirely due to a very favorable binding entropy, while its even higher affinity for Cu+ is due to a favorable binding enthalpy and entropy. The binding thermodn. provide insight into bond enthalpies at the blue copper site and entropic contributions from desolvation and proton displacement. These values were used in thermodn. cycles to det. the enthalpic and entropic contributions to the free energy of redn. and thus the redn. potential. The redn. thermodn. obtained with this method are in good agreement with previous results from temp.-dependent electrochem. measurements. The calorimetry method, however, provides new insight into contributions from the initial (oxidized) and final (reduced) states of the redn. Since ITC measurements quantify the protons that are displaced upon metal binding, the proton transfer that is coupled with electron transfer is also detd. with this method. Preliminary results for Cu2+ and Cu+ binding to the Phe114Pro variant of azurin demonstrate the insight about protein tuning of the redn. potential that is provided by the binding thermodn. of each metal oxidn. state.
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    Konermann, L. Addressing a Common Misconception: Ammonium Acetate as Neutral pH “Buffer” for Native Electrospray Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2017, 28, 1827– 1835,  DOI: 10.1007/s13361-017-1739-3
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    Addressing a Common Misconception: Ammonium Acetate as Neutral pH "Buffer" for Native Electrospray Mass Spectrometry
    Konermann, Lars
    Journal of the American Society for Mass Spectrometry (2017), 28 (9), 1827-1835CODEN: JAMSEF; ISSN:1044-0305. (Springer)
    A review and discussion. Native ESI-MS involves the transfer of intact proteins and biomol. complexes from soln. into the gas phase. One potential pitfall is the occurrence of pH-induced changes that can affect the analyte while it is still surrounded by solvent. Most native ESI-MS studies employ neutral aq. ammonium acetate solns. It is a widely perpetuated misconception that ammonium acetate buffers the analyte soln. at neutral pH. By definition, a buffer consists of a weak acid and its conjugate weak base. The buffering range covers the weak acid pKa ± 1 pH unit. NH4+ and CH3-COO- are not a conjugate acid/base pair, which means that they do not constitute a buffer at pH 7. Dissoln. of ammonium acetate salt in water results in pH 7, but this pH is highly labile. Ammonium acetate does provide buffering around pH 4.75 (the pKa of acetic acid) and around pH 9.25 (the pKa of ammonium). This implies that neutral ammonium acetate solns. electrosprayed in pos. ion mode will likely undergo acidification down to pH 4.75 ± 1 in the ESI plume. Ammonium acetate nonetheless remains a useful additive for native ESI-MS. It is a volatile electrolyte that can mimic the solvation properties experienced by proteins under physiol. conditions. Also, a drop from pH 7 to around pH 4.75 is less dramatic than the acidification that would take place in pure water. It is hoped that the habit of referring to pH 7 solns. as ammonium acetate "buffer" will disappear from the literature. Ammonium acetate "soln." should be used instead.
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    Raamat, E.; Kaupmees, K.; Ovsjannikov, G.; Trummal, A.; Kütt, A.; Saame, J.; Koppel, I.; Kaljurand, I.; Lipping, L.; Rodima, T.; Pihl, V.; Koppel, I. A.; Leito, I. Acidities of strong neutral Brønsted acids in different media. J. Phys. Org. Chem. 2013, 26, 162– 170,  DOI: 10.1002/poc.2946
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    Acidities of strong neutral Bronsted acids in different media
    Raamat, Elin; Kaupmees, Karl; Ovsjannikov, Gea; Trummal, Aleksander; Kuett, Agnes; Saame, Jaan; Koppel, Ivar; Kaljurand, Ivari; Lipping, Lauri; Rodima, Toomas; Pihl, Viljar; Koppel, Ilmar A.; Leito, Ivo
    Journal of Physical Organic Chemistry (2013), 26 (2), 162-170CODEN: JPOCEE; ISSN:0894-3230. (John Wiley & Sons Ltd.)
    Acidities of different families of acids are examd. in media of different phys. and chem. nature: water, acetonitrile (AN), 1,2-dichloroethane (DCE) and the gas phase, with special emphasis on strong acids. Included are OH (carboxylic acids, alcs., and phenols), NH (sulfonamides, imides), and CH (phenylmalononitriles, etc.) acids as well as HCl, HBr, and HI. Dependence of the acidity trends on moving from water to the gas phase on the nature of the acidity center, and the mol. structure are analyzed. The acidity orders are different in water, AN, DCE, and the gas phase. In some cases the differences are dramatic. AN and DCE display similar acidity order in the set of the studied acids. The decisive factor for the behavior of the acids when transferring between different media is the extent of charge delocalization in the anion and the recently introduced weighted av. pos. sigma parameter in spite of its simplicity enables interpretation of the trends in the majority of cases. Copyright © 2012 John Wiley and Sons, Ltd.
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    Wang, H.; Agnes, G. R. Kinetically labile equilibrium shifts induced by the electrospray process. Anal. Chem. 1999, 71, 4166– 4172,  DOI: 10.1021/ac981375u
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    Kinetically Labile Equilibrium Shifts Induced by the Electrospray Process
    Wang, Hongjun; Agnes, George R.
    Analytical Chemistry (1999), 71 (19), 4166-4172CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    The complexation reactions between the alk. earth metal ions and EDTA were studied by electrospray mass spectrometry to measure the change in concn. of the metal ion-EDTA complex (MY2-) in the gas phase relative to the soln.-phase equil. concn. This work focused on the soln. pH range from 4 to 7 where there exists free metal ions in soln. at equil. The equil. shift, measured through quantitation of the increased abundance of the MY2- species in the gas phase, was largest for barium and smallest for magnesium. The cause of the net equil. shift of the MY2- species is the combined effect of an electrolytic increase in pH within the capillary plus an addnl. shift within the evapg. droplets. In a thin diffusion-limited layer created by the products of electrolysis mixing with the bulk soln. at the ES capillary tip, the labile species reequilibrate at a new, higher pH. In the evapg. droplets, the formation of new labile species due to increased solute concns. is kinetically controlled because the ion residence time in the droplet prior to desorption is only ∼5 μs. These results are briefly discussed with respect to the potential for utilizing electrospray mass spectrometry for kinetically labile equil. studies.
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    McDonald, L. W.; Campbell, J. A.; Clark, S. B. Failure of ESI spectra to represent metal-complex solution composition: A study of lanthanide-carboxylate complexes. Anal. Chem. 2014, 86, 1023– 1029,  DOI: 10.1021/ac401751r
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    Failure of ESI Spectra to Represent Metal-Complex Solution Composition: A Study of Lanthanide-Carboxylate Complexes
    McDonald, Luther W.; Campbell, James A.; Clark, Sue B.
    Analytical Chemistry (Washington, DC, United States) (2014), 86 (2), 1023-1029CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Electrospray ionization-mass spectrometry (ESI-MS) shows great promise as a rapid method to identify metal-ligand complexes in soln. However, its application for quant. detn. of the distribution of species present in complicated equil. is still in its infancy, and a direct correlation between ions obsd. in the gas phase and species expected in soln. must be made with caution. The present work focuses on a seemingly simple system; the complexation of lanthanide cations with the acetate ligand. Using a high resoln. quadrupole time-of-flight mass spectrometer, ions created by electrospray of solns. contg. trivalent Nd and acetate were identified. The gas phase distribution of species was compared to the soln. phase speciation predicted using thermodn. complexation consts. Apparent gas phase speciation diagrams were constructed as a function of soln. conditions and fragmentation potential. Despite the expected variability of metal-ligand complexes as soln. conditions change, the obsd. gas phase speciation was independent of the metal to ligand ratio but dependent on the operating conditions of the ESI-MS.
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    Zhang, M.; Gumerov, D. R.; Kaltashov, I. A.; Mason, A. B. Indirect detection of protein-metal binding: Interaction of serum transferrin with In3+ and Bi3+. J. Am. Soc. Mass Spectrom. 2004, 15, 1658– 1664,  DOI: 10.1016/j.jasms.2004.08.009
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    Indirect detection of protein-Metal binding: Interaction of serum transferrin with In3+ and Bi3+
    Zhang, Mingxuan; Gumerov, Dmitry R.; Kaltashov, Igor A.; Mason, Anne B.
    Journal of the American Society for Mass Spectrometry (2004), 15 (11), 1658-1664CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)
    Transferrins comprise a class of monomeric glycoproteins found in all vertebrates, whose function is iron sequestration and transport. In addn. to iron, serum transferrin also binds a variety of other metals and is believed to provide a route for the in vivo delivery of such metals to cells. In the present study, ESI MS is used to investigate interactions between human serum transferrin and two nonferrous metals, indium (a commonly used imaging agent) and bismuth (a component of many antiulcer drugs). While the UV-Vis absorption spectroscopy measurements clearly indicate that both metals bind strongly to transferrin in soln., the metal-protein complex can be detected by ESI MS only for indium, but not for bismuth. Despite the apparently low stability of the transferrin-bismuth complex in the gas phase, presence of such complex in soln. can be established by ESI MS indirectly. This is done by monitoring the evolution of charge state distributions of transferrin ions upon acid-induced protein unfolding in the presence and in the absence of the metal in soln. The anomalous instability of the transferrin-bismuth complex in the gas phase is rationalized in terms of conformational differences between this form of transferrin and the holo-forms of this protein produced by binding of metals with smaller ionic radii (e.g., Fe3+ and In3+). The large size of Bi3+ ion is likely to prevent formation of a closed conformation (canonical structure of the holo-protein), resulting in a non-native metal coordination. It is suggested that transferrin retains the open conformation (characteristic of the apo-form) upon binding Bi3+, with only two ligands in the metal coordination sphere provided by the protein itself. This suggestion is corroborated by the results of CD measurements in the near-UV range. Since the cellular consumption of metals in the transferrin cycle critically depends upon recognition of the holo-protein complex by the transferrin receptor, the noncanonical conformation of the transferrin-bismuth complex may explain very inefficient delivery of bismuth to cells even when a high dosage of bismuth-contg. drugs is administered for prolonged periods of time.
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    Gatlin, C. L.; Turecek, F.; Vaisar, T. Determination of soluble Cu (I) and Cu (II) species in jet fuel by electrospray ionization mass spectrometry. Anal. Chem. 1994, 66, 3950– 3958,  DOI: 10.1021/ac00094a016
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    Determination of Soluble Cu(I) and Cu(II) Species in Jet Fuel by Electrospray Ionization Mass Spectrometry
    Gatlin, Christine L.; Turecek, Frantisek; Vaisar, Tomas
    Analytical Chemistry (1994), 66 (22), 3950-8CODEN: ANCHAM; ISSN:0003-2700.
    Sol. Cu(I) and Cu(II) species in jet fuel (e.g., formed by exposure of jet fuels to Cu surfaces and extd. by metal deactivator additives) were analyzed either by electrospray-ionization mass spectrometry combined with online liq. chromatog. on poly(vinyl alc.) sorbent or by off-line solvent extn. Detection limits in the 30-150 ppb range were achieved for Cu2+DMD (Cu2+ complex with N,N'-disalicylidene-1,2-propylenediamine), which was detected by mass spectrometry as its protonated ion. Low ppb detection limits were possible for the Na+ adduct. Collisional activation of Cu2+ carboxylate complexes in the gas phase induces intramol. electron transfer, resulting in redn. of the metal ion to Cu+ with concomitant ligand oxidn. and elimination.
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    Lavanant, H.; Hoppilliard, Y. Formation and fragmentation of α-amino acids complexed by Cu+. J. Mass Spectrom. 1997, 32, 1037– 1049,  DOI: 10.1002/(SICI)1096-9888(199711)32:10<1037::AID-JMS556>3.0.CO;2-L
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    Formation and fragmentation of α-amino acids complexed by Cu+
    Lavanant, Helene; Hoppilliard, Yannik
    Journal of Mass Spectrometry (1997), 32 (10), 1037-1049CODEN: JMSPFJ; ISSN:1076-5174. (Wiley)
    Gas phase complexes of α-amino acids with Cu+ have been engendered in a plasma desorption ion source by bombarding a mixt. of α-amino acids and cupric salts. The resulting adducts MCu+ and the fragments that include copper were studied using a time-of-flight analyzer. The main fragments result from a loss of 46 u, this fragmentation being very similar to the one obsd. for protonated mols. One noticeable exception, however, is arginine and lysine which give very little loss of 46 u as protonated mols. but do give a significant amt. of it when cationized by Cu+. The study of valine-d, has shown that the migration accompanying the fragmentations principally involves the hydrogens linked to heteroatoms. The deuteration of exchangeable hydrogens, done on several amino acids, confirmed these results. This fact, plus evidence of an interaction between Cu+ and the side chain of non-aliph. α-amino acids, has led to the suggestion of several mechanisms.
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    Lavanant, H.; Virelizier, H.; Hoppilliard, Y. Reduction of copper (II) complexes by electron capture in an electrospray ionization source. J. Am. Soc. Mass Spectrom. 1998, 9, 1217– 1221,  DOI: 10.1016/S1044-0305(98)00100-7
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    Reduction of copper(II) complexes by electron capture in an electrospray ionization source
    Lavanant, Helene; Virelizier, Henri; Hoppilliard, Yannik
    Journal of the American Society for Mass Spectrometry (1998), 9 (11), 1217-1221CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Science Inc.)
    The relative proportion of 1:1 Cu(I)- and Cu(II)-peptide complexes, PeptCu(I)+ and [Pept - H+Cu(II)]+, yielded by electrospray ionization of copper sulfate and peptide, H-Gly-His-Lys-OH, solns. in H2O/MeOH was examd. under different source conditions. Two factors leading to an increase in Cu(I):complex ratio were found. First, the increase of nozzle-skimmer voltages caused collision-induced dissocn. of Cu(II) complexes, and most probably, this favored ligand-to-metal electron transfers that resulted in the decoordination of oxydated ligands to form PeptCu+. Second, independent of these "inner sphere" processes that involve only electron exchange inside the coordination sphere around the metal cation, an increase in source voltages with a concomitant increase of current and, supposedly, electron counterflow between the counterelectrode and the capillary caused an increase in PeptCu+ relative proportion. The hypothesis that an "outer sphere" electron capture might happen in these conditions was verified by using discharge suppressing SF6 gas as nebulizing gas. The electroneg. gas reduced the current brought on by high voltages and inhibited the PeptCu+ increase phenomenon.
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    Tsybizova, A.; Roithová, J. Copper-catalyzed reactions: Research in the gas phase. Mass Spectrom. Rev. 2016, 35, 85– 110,  DOI: 10.1002/mas.21464
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    Copper-catalyzed reactions: Research in the gas phase
    Tsybizova, Alexandra; Roithova, Jana
    Mass Spectrometry Reviews (2016), 35 (1), 85-110CODEN: MSRVD3; ISSN:0277-7037. (John Wiley & Sons, Inc.)
    Electrospray ionization mass spectrometry (ESI-MS) is becoming an important tool for mechanistic studies in org. and organometallic chem. It allows investigation of reaction mixts. including monitoring of reactants, products, and intermediates, studying properties of the intermediates and their reactivity. Studying the reactive species in the gas phase can be advantageously combined with theor. calcns. This review is focused on ESI-MS studies of copper-catalyzed reactions. Possible effects of the electrospray process on the transfer of the copper complexes to the gas phase are discussed. The plethora of mass spectrometric approaches is demonstrated on copper mediated C-H activations, cross coupling reactions, rearrangements, organocuprate chem., and other examples.
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    Di Marco, V. B.; Bombi, G. G.; Zambon, S.; Traldi, P. Metal-ligand solution equilibria studied by electrospray ionization mass spectrometry: Effect of instrumental parameters. J. Mass Spectrom. 2009, 44, 120– 127,  DOI: 10.1002/jms.1481
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    Metal-ligand solution equilibria studied by electrospray ionization mass spectrometry: effect of instrumental parameters
    Di Marco, Valerio B.; Bombi, G. Giorgio; Zambon, Stefano; Traldi, Pietro
    Journal of Mass Spectrometry (2009), 44 (1), 120-127CODEN: JMSPFJ; ISSN:1076-5174. (John Wiley & Sons Ltd.)
    Electrospray ionization mass spectrometry (ESI-MS) is being increasingly employed in the study of metal-ligand equil. in aq. soln. In the present work, the ESI-MS spectral changes due to different settings of the following instrumental parameters are analyzed: the soln. flow rate (FS), the nebulizer gas flow rate (FG), the sprayer potential (E), and the temp. of the entrance capillary (T). Twenty-eight spectra were obtained for each of six samples contg. aluminum(III) and 2,3-dihydroxypyridine at various pH, in the absence or in the presence of a buffer and of sodium ions. Among the considered instrumental parameters, T produced the largest effects on the ionic intensities. FS and FG affected the ESI-MS spectra to a lower extent than T. In the investigated conditions E had the weakest effects on the spectra. The correlations obsd. between the ionic intensities and these instrumental parameters were interpreted considering the presence of three kinds of perturbations occurring in the ESI-MS ion source: formation of some dimers in the droplets, different transfer efficiencies from the droplets to the gas phase for different complexes (according to their surface activity), and subsequent partial thermal decompn. of the dimers and of one of the monomeric complexes in the gas phase. The results obtained show that the evaluation of the effects produced in the ESI-MS spectra by a change of instrumental parameters can allow to identify the perturbations occurring when metal-ligand solns. are studied by ESI-MS.
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    Kotuniak, R.; Frączyk, T.; Skrobecki, P.; Płonka, D.; Bal, W. Gly-His-Thr-Asp-Amide, an Insulin-Activating Peptide from the Human Pancreas Is a Strong Cu(II) but a Weak Zn(II) Chelator. Inorg. Chem. 2018, 57, 15507– 15516,  DOI: 10.1021/acs.inorgchem.8b02841
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    Gly-His-Thr-Asp-Amide, an Insulin-Activating Peptide from the Human Pancreas Is a Strong Cu(II) but a Weak Zn(II) Chelator
    Kotuniak, Radoslaw; Fraczyk, Tomasz; Skrobecki, Piotr; Plonka, Dawid; Bal, Wojciech
    Inorganic Chemistry (2018), 57 (24), 15507-15516CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
    The Cu(II) and Zn(II) binding abilities of Gly-His-Thr-Asp-amide (GHTD-am), a tetrapeptide co-released from the pancreas along with insulin, were studied using UV-vis and CD spectroscopies, potentiometry, and calorimetry. GHTD-am is a very strong Cu(II) chelator, forming a three-nitrogen complex with a conditional affinity const. CK at pH 7.4 of 4.5 × 1012 M-1. The fourth coordination site can be occupied by a solvent mol. or a ternary ligand, such as imidazole, with CK on the order of several hundred reciprocal molar. The Zn(II) binding ability of GHTD-am is relatively weak, with CK values at pH 7.4 of 3.0 × 104 and 2.0 × 103 M-1 for the first and second GHTD-am mol. coordinated, resp. These results are discussed in light of the modes of interactions of Zn(II) and Cu(II) ions with insulin. A direct effect of GHTD-am on the Zn(II) interactions with insulin is unlikely, but its Cu(II) complex may have a biol. relevance because of its high affinity and ability to form ternary complexes.
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    Płonka, D.; Bal, W. The N-terminus of hepcidin is a strong and potentially biologically relevant Cu(II) chelator. Inorg. Chim. Acta 2018, 472, 76– 81,  DOI: 10.1016/j.ica.2017.06.051
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    The N-terminus of hepcidin is a strong and potentially biologically relevant Cu(II) chelator
    Plonka, Dawid; Bal, Wojciech
    Inorganica Chimica Acta (2018), 472 (), 76-81CODEN: ICHAA3; ISSN:0020-1693. (Elsevier B.V.)
    Hepcidin is an iron regulatory hormone, involved also in immune response in vertebrates. It contains the N-terminal Asp-Thr-His site able to bind Cu(II) ions. A significant discrepancy exist in the literature regarding Cu(II) affinity of this site in hepcidin. Our study focused on the model DTHFPI-NH2 hexapeptide reflecting the N-terminal motif of mature hepcidin. Using potentiometry, UV-vis and CD spectroscopies we demonstrated that DTHFPI-NH2 is the strongest Cu(II) binding peptide discovered so far. A competition assay with human serum albumin (HSA) confirmed this high affinity and demonstrated that DTHFPI-NH2. withdraws all Cu(II) from HSA under equimolar concns. Based on these results we propose that hepcidin could exist as Cu(II) complex in blood, esp. under inflammatory conditions, when its serum concn. is elevated.
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    Bossak, K.; Drew, S. C.; Stefaniak, E.; Płonka, D.; Bonna, A.; Bal, W. The Cu(II) affinity of the N-terminus of human copper transporter CTR1: Comparison of human and mouse sequences. J. Inorg. Biochem. 2018, 182, 230– 237,  DOI: 10.1016/j.jinorgbio.2018.01.011
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    The Cu(II) affinity of the N-terminus of human copper transporter CTR1: Comparison of human and mouse sequences
    Bossak, Karolina; Drew, Simon C.; Stefaniak, Ewelina; Plonka, Dawid; Bonna, Arkadiusz; Bal, Wojciech
    Journal of Inorganic Biochemistry (2018), 182 (), 230-237CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)
    Copper Transporter 1 (CTR1) is a homotrimeric membrane protein providing the main route of copper transport into eukaryotic cells from the extracellular milieu. Its N-terminal extracellular domain, rich in His and Met residues, is considered responsible for directing copper into the transmembrane channel. Most of vertebrate CTR1 proteins contain the His residue in position three from N-terminus, creating a well-known Amino Terminal Cu(II)- and Ni(II)-Binding (ATCUN) site. CTR1 from humans, primates and many other species contains the Met-Asp-His (MDH) sequence, while some rodents including mouse have the Met-Asn-His (MNH) N-terminal sequence. CTR1 is thought to collect Cu(II) ions from blood copper transport proteins, including albumin, but previous reports indicated that the affinity of N-terminal peptide/domain of CTR1 is significantly lower than that of albumin, casting serious doubt on this aspect of CTR1 function. Using potentiometry and spectroscopic techniques we demonstrated that MDH-amide, a tripeptide model of human CTR1 N-terminus, binds Cu(II) with K of 1.3 × 1013 M-1 at pH 7.4, ∼13 times stronger than Human Serum Albumin (HSA), and MNH-amide is even stronger, K of 3.2 × 1014 M-1 at pH 7.4. These results indicate that the N-terminus of CTR1 may serve as intermediate binding site during Cu(II) transfer from blood copper carriers to the transporter. MDH-amide, but not MNH-amide also forms a low abundance complex with non-ATCUN coordination involving the Met amine, His imidazole and Asp carboxylate. This species might assist Cu(II) relay down the peptide chain or its redn. to Cu(I), both steps necessary for the CTR1 function.
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    Bossak-Ahmad, K.; Mital, M.; Płonka, D.; Drew, S. C.; Bal, W. Oligopeptides Generated by Neprilysin Degradation of β-Amyloid Have the Highest Cu(II) Affinity in the Whole Aβ Family. Inorg. Chem. 2019, 58, 932– 943,  DOI: 10.1021/acs.inorgchem.8b03051
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    Oligopeptides generated by neprilysin degradation of β-amyloid have the highest Cu(II) affinity in the whole Aβ Family
    Bossak-Ahmad, Karolina; Mital, Mariusz; Plonka, Dawid; Drew, Simon C.; Bal, Wojciech
    Inorganic Chemistry (2019), 58 (1), 932-943CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
    The catabolism of β-amyloid (Aβ) is carried out by numerous endopeptidases including neprilysin, which hydrolyzes peptide bonds preceding positions 4, 10, and 12 to yield Aβ4-9 and a minor Aβ12-x species. Alternative processing of the amyloid precursor protein by β-secretase also generates the Aβ11-x species. All these peptides contain a Xxx-Yyy-His sequence, also known as an ATCUN or NTS motif, making them strong chelators of Cu(II) ions. We synthesized the corresponding peptides, Phe-Arg-His-Asp-Ser-Gly-OH (Aβ4-9), Glu-Val-His-His-Gln-Lys-am (Aβ11-16), Val-His-His-Gln-Lys-am (Aβ12-16), and pGlu-Val-His-His-Gln-Lys-am (pAβ11-16), and investigated their Cu(II) binding properties using potentiometry, and UV-vis, CD, and ESR spectroscopies. We found that the three peptides with unmodified N-termini formed square-planar Cu(II) complexes at pH 7.4 with analogous geometries but significantly varied Kd values of 6.6 fM (Aβ4-9), 9.5 fM (Aβ12-16), and 1.8 pM (Aβ11-16). Cyclization of the N-terminal Glu11 residue to the pyroglutamate species pAβ11-16 dramatically reduced the affinity (5.8 nM). The Cu(II) affinities of Aβ4-9 and Aβ12-16 are the highest among the Cu(II) complexes of Aβ peptides. Using fluorescence spectroscopy, we demonstrated that the Cu(II) exchange between the Phe-Arg-His and Val-His-His motifs is very slow, on the order of days. These results are discussed in terms of the relevance of Aβ4-9, a major Cu(II) binding Aβ fragment generated by neprilysin, as a possible Cu(II) carrier in the brain.
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    Young, T. R.; Xiao, Z. Principles and practice of determining metal-protein affinities. Biochem. J. 2021, 478, 1085– 1116,  DOI: 10.1042/BCJ20200838
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    Principles and practice of determining metal-protein affinities
    Young, Tessa R.; Xiao, Zhiguang
    Biochemical Journal (2021), 478 (5), 1085-1116CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
    A review. Metal ions play many crit. roles in biol., as structural and catalytic cofactors, and as cell regulatory and signalling elements. The metal-protein affinity, expressed conveniently by the metal dissocn. const., KD, describes the thermodn. strength of a metal-protein interaction and is a key parameter that can be used, for example, to understand how proteins may acquire metals in a cell and to identify dynamic elements (e.g. cofactor binding, changing metal availabilities) which regulate protein metalation in vivo. Here, we outline the fundamental principles and practical considerations that are key to the reliable quantification of metal-protein affinities. We review a selection of spectroscopic probes which can be used to det. protein affinities for essential biol. transition metals (including Mn(II), Fe(II), Co(II), Ni(II), Cu(I), Cu(II) and Zn(II)) and, using selected examples, demonstrate how rational probe selection combined with prudent exptl. design can be applied to det. accurate KD values.
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    Sokołowska, M.; Bal, W. Cu(II) complexation by “non-coordinating” N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES buffer). J. Inorg. Biochem. 2005, 99, 1653– 1660,  DOI: 10.1016/j.jinorgbio.2005.05.007
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    Cu(II) complexation by "non-coordinating" N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES buffer)
    Sokolowska, Magdalena; Bal, Wojciech
    Journal of Inorganic Biochemistry (2005), 99 (8), 1653-1660CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier B.V.)
    The combined potentiometric and spectroscopic studies of interactions of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) with Cu(II) demonstrated that this popular buffer, commonly labeled as noncoordinating forms a CuL+ complex, with the log βCuL value of 3.22. This complex undergoes alk. hydrolysis above pH 6, resulting in Cu(OH)2 pptn. However, the presence of HEPES at a typical concn. of 100 mM at pH 7.4 elevates the apparent binding const., being detd. for a complex of another ligand, by a factor of 80. HEPES does not form ternary complexes with amino acids Ala, Trp, and His, but may do so with other bioligands, such as nucleotides. Therefore, HEPES can still be recommended for Cu(II) studies in place of other common buffers, such as Tris and phosphate, but appropriate corrections and precautions should be applied in quant. expts.
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    Krężel, A.; Wójcik, J.; Maciejczyk, M.; Bal, W. May GSH and L-His contribute to intracellular binding of zinc? Thermodynamic and solution structural study of a ternary complex. Chem. Commun. 2003, 704– 705,  DOI: 10.1039/b300632h
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    May GSH and L-His contribute to intracellular binding of zinc? Thermodynamic and solution structural study of a ternary complex
    Krezel, Artur; Wojcik, Jacek; Maciejczyk, Maciej; Bal, Wojciech
    Chemical Communications (Cambridge, United Kingdom) (2003), (6), 704-705CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    GSH and L-His are abundant biomols. and likely biol. ligands for Zn(II) under certain conditions. Potentiometric titrns. provide evidence of formation of ternary Zn(II) complexes with GSH and L-His or D-His with slight stereoselectivity in favor of L-His (ca. 1 log unit of stability const.). The soln. structure of the ZnH(GSH)(L-His)(H2O) complex at pH 6.8, detd. by NMR, includes tridentate L-His, monodentate (sulfur) GSH, and weak interligand interactions. Calcns. of competitiveness of this complex for Zn(II) binding at pH 7.4 indicate that it is likely to be formed in vivo under conditions of GSH depletion. Otherwise, GSH alone emerges as a likely Zn(I) carrier.
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    Jeżowska-Bojczuk, M.; Kaczmarek, P.; Bal, W.; Kasprzak, K. S. Coordination mode and oxidation susceptibility of nickel(II) complexes with 2’-deoxyguanosine 5′-monophosphate and L-histidine. J. Inorg. Biochem. 2004, 98, 1770– 1777,  DOI: 10.1016/j.jinorgbio.2004.08.002
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    Coordination mode and oxidation susceptibility of nickel(II) complexes with 2'-deoxyguanosine 5'-monophosphate and L-histidine
    Jezowska-Bojczuk, Malgorzata; Kaczmarek, Piotr; Bal, Wojciech; Kasprzak, Kazimierz S.
    Journal of Inorganic Biochemistry (2004), 98 (11), 1770-1777CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier B.V.)
    The formation of binary and ternary complexes of Ni(II) with two biol. relevant mols., 2'-deoxyguanosine 5'-monophosphate (dGMP) and L-histidine (histidine or His) was characterized by potentiometry and UV-visible spectroscopy. For dGMP, the mononuclear complexes with stoichiometries NiH2L+, NiHL and NiL- were found. In the mixed system the ternary complexes NiH2LA, NiHLA- and NiLA2- were detected. In binary systems, the Ni(II) ion coordinates to dGMP through the N-7 atom of its purine ring and indirectly through a water mol. bonded to the phosphate group, while in ternary complexes Ni(II) is bonded to all three histidine donors and directly to the phosphate group of dGMP. Both binary and ternary complexes are susceptible to oxidn. by H2O2, with the increased formation of 8-oxo-dGMP in the ternary system. The toxicol. relevance of these findings stems from possible disturbance by the major biol. Ni(II)-His complex of the nucleotide pools homeostasis through the formation of ternary species and oxidn. promotion, as well as from 8-oxo-dGMP capacity to inhibit enzymic elimination of pro-mutagenic oxidized nucleotides from such pools.
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    Sigel, H.; Martin, R. B. Coordinating properties of the amide bond. Stability and structure of metal ion complexes of peptides and related ligands. Chem. Rev. 1982, 82, 385– 426,  DOI: 10.1021/cr00050a003
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    Coordinating properties of the amide bond. Stability and structure of metal ion complexes of peptides and related ligands
    Sigel, Helmut; Martin, R. Bruce
    Chemical Reviews (Washington, DC, United States) (1982), 82 (4), 385-426CODEN: CHREAY; ISSN:0009-2665.
    A review with 409 refs.
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    Wilcox, D. E. Isothermal titration calorimetry of metal ions binding to proteins: An overview of recent studies. Inorg. Chim. Acta 2008, 361, 857– 867,  DOI: 10.1016/j.ica.2007.10.032
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    Isothermal titration calorimetry of metal ions binding to proteins: An overview of recent studies
    Wilcox, Dean E.
    Inorganica Chimica Acta (2008), 361 (4), 857-867CODEN: ICHAA3; ISSN:0020-1693. (Elsevier B.V.)
    A review. Isothermal titrn. calorimetry (ITC) is a technique that is capable of quantifying the stoichiometry, equil. consts. and thermodn. of mol. binding events. Thus, important information about the interaction of metal ions with biol. macromols. can be obtained with ITC measurements. This review highlights many of the recent studies of metal ions binding to proteins that have used ITC to quantify the thermodn. of metal-protein interactions.
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    Kostyukevich, Y.; Kononikhin, A.; Popov, I.; Indeykina, M.; Kozin, S. A.; Makarov, A. A.; Nikolaev, E. Supermetallization of peptides and proteins during electrospray ionization. J. Mass Spectrom. 2015, 50, 1079– 1087,  DOI: 10.1002/jms.3622
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    Supermetallization of peptides and proteins during electrospray ionization
    Kostyukevich, Yury; Kononikhin, Alexey; Popov, Igor; Indeykina, Maria; Kozin, Sergey A.; Makarov, Alexander A.; Nikolaev, Eugene
    Journal of Mass Spectrometry (2015), 50 (9), 1079-1087CODEN: JMSPFJ; ISSN:1076-5174. (John Wiley & Sons Ltd.)
    The formation of metal-peptide complexes during electrospray ionization (ESI) is a widely known phenomenon and is often considered to be undesirable. Such effect considerably limits the use of ESI mass spectrometry for the study of biol. relevant metal-peptide compds. that are present in the soln. and play crit. roles in many bioprocesses such as progression of neurodegenerative diseases. Under specific conditions such as high temp. of the desolvating capillary, an interesting effect, which can be called supermetallization, occurs. Using a model peptide Aβ amyloid domain 1-16, an increase in the temp. of the desolvating capillary results in multiple substitutions of hydrogen atoms by Zn atoms in this peptide. At high temps. (T ∼ 400°), up to 11 zinc atoms can be covalently bound to (1-16) Aβ. Supermetallization of (1-16) Aβ depends on the solvent compn. and pH. Supermetallization was also demonstrated for proteins, such as ubiquitin and cytochrome C. That proves that the supermetallization is a general phenomenon for peptides and proteins. For the structural study of supermetallized complexes, electron-capture dissocn. (ECD) fragmentation was applied. The effect of hydrogen rearranging during ECD was obsd. In addn., quantum chem. calcns. were used to est. the possible structures of different supermetallized complexes. These results allow a more deep understanding of the limitations of the use of ESI mass spectrometry for the study of biol. relevant metal-peptide complexes.
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    Chelate Effect and Thermodynamics of Metal Complex Formation in Solution: A Quantum Chemical Study
    Vallet, Valerie; Wahlgren, Ulf; Grenthe, Ingmar
    Journal of the American Chemical Society (2003), 125 (48), 14941-14950CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The accuracy of quantum chem. predictions of structures and thermodn. data for metal complexes depends both on the quantum chem. methods and the chem. models used. A thermodn. analog of the Eigen-Wilkins mechanism for ligand substitution reactions (Model A) turns out to be sufficiently simple to catch the essential chem. of complex formation reactions and allows quantum chem. calcns. at the ab initio level of thermodn. quantities both in gas phase and soln.; the latter by using the conductor-like polarizable continuum (CPCM) model. Model A describes the complex formation as a two-step reaction: [M(H2O)x](aq) + L(aq) .dblharw. [M(H2O)x],L(aq); and [M(H2O)x],L(aq) .dblharw. [M(H2O)x-1L],(H2O)(aq). The first step, the formation of an outer-sphere complex is described using the Fuoss equation and the second, the intramol. exchange between an entering ligand from the second and water in the first coordination shell, using quantum chem. methods. The thermodn. quantities for this model were compared to those for the reaction: [M(H2O)x](aq) + L(aq) .dblharw. [M(H2O)x-1L](aq) + (H2O)(aq) (Model B), as calcd. for each reactant and product sep. The models were tested using complex formation between Zn2+ and ammonia, methylamine, and ethylenediamine, and complex formation and chelate ring closure reactions in binary and ternary UO22+-oxalate systems. The results show that the Gibbs energy of reaction for Model A are not strongly dependent on the no. of water ligands and the structure of the second coordination sphere; it provides a much more precise est. of the thermodn. of complex formation reactions in soln. than that obtained from Model B. The agreement between the exptl. and calcd. data for the formation of Zn(NH3)2+(aq) and Zn(NH3)2+2(aq) is better than 8 kJ/mol for the former, as compared to 30 kJ/mol or larger, for the latter. The Gibbs energy of reaction obtained for the UO2+2 oxalate systems using model B differs between 80 and 130 kJ/mol from the exptl. results, whereas the agreement with Model A is better. The errors in the quantum chem. ests. of the entropy and enthalpy of reaction are somewhat larger than those for the Gibbs energy, but still in fair agreement with expts.; adding water mols. in the second coordination sphere improves the agreement significantly. Reasons for the different performance of the two models are discussed. The quantum chem. data were used to discuss the microscopic basis of exptl. enthalpy and entropy data, to det. the enthalpy and entropy contributions in chelate ring closure reactions and to discuss the origin of the so-called "chelate effect". Contrary to many earlier suggestions, this is not even in the gas phase, a result of changes in translation entropy contributions. There is no simple explanation of the high stability of chelate complexes; it is a result of both enthalpy and entropy contributions that vary from one system to the other.
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  1. Peter J. H. Williams, Ian C. Chagunda, J. Scott McIndoe. OptiMS: An Accessible Program for Automating Mass Spectrometry Parameter Optimization and Configuration. Journal of the American Society for Mass Spectrometry 2024, 35 (3) , 449-455. https://doi.org/10.1021/jasms.3c00354
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  9. Radosław Kotuniak, Wojciech Bal. Kinetics of Cu( ii ) complexation by ATCUN/NTS and related peptides: a gold mine of novel ideas for copper biology. Dalton Transactions 2021, 51 (1) , 14-26. https://doi.org/10.1039/D1DT02878B
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  • Abstract

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    Figure 1

    Figure 1. Structures of Cu(II) complexes at the N-terminal site of peptides having His in the 2nd (B) or 3rd position (A) and (C). R(n) mark side chains and the remainder of the peptide chain.

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    Figure 2

    Figure 2. Theoretical dissociation curves of Cu(II)/peptide 1:1 complexes at pH 7.4.

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    Figure 3

    Figure 3. Experimental and theoretical binding isotherms for Cu(II)/Aβ4-16 (A) and Cu(II)/hepc6 (B) 100 nM 1:1 complexes titrated with histidine as a weak competitor. Dotted lines mark concentration at which 50% of the peptide is occupied with Cu(II) ion.

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    Figure 4

    Figure 4. pH-dependent species distribution of Cu(II) complexes of hepc6 (top) and GGH (bottom) calculated for 1 mM peptides and 0.8 mM Cu(II) based on potentiometric data from ref (65) and ref (21), respectively.

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    Figure 5

    Figure 5. Prediction of pH values in electrospray stipulated by the literature log CK values compared with the values calculated from serial dilution ESI-MS (data from Table 1). Solid curves represent the pH dependence of literature log CK values for individual peptides. Circles mark the ESI-MS-derived log Kd values ± SD placed along these curves to indicate the pH of spray droplets expected if acidification were the sole source of the observed Kd decrease. Gray area marks the buffering zone of ammonium acetate (pH 4.75 ± 1).

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    Figure 6

    Figure 6. Stopped-flow spectra of Cu(II)-hepc6 in 20 mM ammonium acetate initially at pH 7.4, subjected to rapid pH decrease by mixing with concentrated acetic acid. Color-coded spectra were recorded every 1.5 ms with the instrument dead time of 2 ms. (A) First 11 ms of the reaction and (B) all spectra recorded until equilibrium was reached. The band at 525 nm represents the fully formed (4N) ATCUN complex, stable at pH 7.4, and the band at 750 nm represents the intermediate (2N) complex. (20)

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    Figure 7

    Figure 7. Kinetic traces at 525 nm for pH–jump experiments on Cu(II)-hepc6 presented in Figure 6 for pH 3 (A), 4 (B), and 5 (C). Left: full traces recorded over 1.5 s and 1st order kinetic fits (red lines). Right: the first 30 ms of traces. Vertical lines mark the 0 ms (reaction start) and the 10 ms time points.

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    Figure 8

    Figure 8. Experimental and theoretical binding isotherms of Cu/Aβ4-16 (A) and Cu/hepc6 (B) 1:1 complexes titrated with histidine as weak competitor.

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  • References

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      The tripeptide H-Gly-His-Lys-OH (GHL) [49557-75-7] is a human plasma constituent which modulates the growth and viability of a variety of cell types and organisms. GHL is complexed with transition metal ions Cu2+ and Fe2+ in vivo and may exert its biol. effects as a peptide-metal chelate. At physiol. pH in vitro, GHL assocs. with ionic Cu, Co, Fe, Mo, Mn, Ni, and Zn,but has no affinity for Ca, Mg, K, and Na. GHL acted synergistically with Cu, Fe, Co, and Zn to alter patterns of cell growth in monolayer cultures of a tumorigenic hepatoma cell line (HTC4). These transition metals induced cellular flattening and adhesion to support surfaces, and inhibited DNA synthesis and lactic acid prodn. when growth was limited by redn. of serum concns. in medium. These inhibitory effects were neutralized, and intercellular adhesion and growth were stimulated by GHL in medium at nanomolar concns. Cu and Fe were the most active metals when combined with GHL. Perhaps the inability of HTC4 cultures to replicate without adequate concns. of serum in medium reflects a deficiency of GHL and transition metals, which appear to form complexes prior to interaction with cells. The obsd. effects of GHL-metal complexes, including stimulation of cellular adhesiveness to substratum (flattening) and intercellular attachment (monolayer formation), appear to satisfy requirements for growth of hepatoma cells in monolayer culture.
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      A review. The human peptide GHK (glycyl-L-histidyl-L-lysine) has multiple biol. actions, all of which, according to our current knowledge, appear to be health pos. It stimulates blood vessel and nerve outgrowth, increases collagen, elastin, and glycosaminoglycan synthesis, as well as supports the function of dermal fibroblasts. GHK's ability to improve tissue repair has been demonstrated for skin, lung connective tissue, boney tissue, liver, and stomach lining. GHK has also been found to possess powerful cell protective actions, such as multiple anti-cancer activities and anti-inflammatory actions, lung protection and restoration of chronic obstructive pulmonary disease (COPD) fibroblasts, suppression of mols. thought to accelerate the diseases of aging such as NFB, anti-anxiety, anti-pain and anti-aggression activities, DNA repair, and activation of cell cleansing via the proteasome system. Recent genetic data may explain such diverse protective and healing actions of one mol., revealing multiple biochem. pathways regulated by GHK.
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      Purification and partial characterization of α factor, a mating type specific inhibitor of cell reproduction from Saccharomyces cerevisiae
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      European Journal of Biochemistry (1973), 35 (2), 357-65CODEN: EJBCAI; ISSN:0014-2956.
      Cells of S. cerevisiae exhibiting the α mating type excreted into the culture medium a low-mol.-wt. substance, termed α factor. This factor, which specifically inhibited DNA replication in cells of the opposite mating type a, was purified >100,000-fold from culture filtrates of α cells. Purified α factor appeared to be homogeneous as judged from thin-layer chromatog. and thin-layer electrophoresis. Leucine, glycine, proline, glutamic acid, tyrosine, tryptophan, and possibly histidine were identified in the factor. In addn., purified α factor contained Cu2+. Gel filtration on Sephadex G-25 in 8M urea indicated a mol. wt. in the range of 1400. The properties of the purified α factor are consistent with those of a low-mol.-wt. peptide.
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      Bossak, K.; Mital, M.; Poznański, J.; Bonna, A.; Drew, S.; Bal, W. Interactions of α-Factor-1, a Yeast Pheromone, and Its Analogue with Copper(II) Ions and Low-Molecular-Weight Ligands Yield Very Stable Complexes. Inorg. Chem. 2016, 55, 7829– 7831,  DOI: 10.1021/acs.inorgchem.6b01441
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      Interactions of α-Factor-1, a Yeast Pheromone, and Its Analogue with Copper(II) Ions and Low-Molecular-Weight Ligands Yield Very Stable Complexes
      Bossak, Karolina; Mital, Mariusz; Poznanski, Jaroslaw; Bonna, Arkadiusz; Drew, Simon; Bal, Wojciech
      Inorganic Chemistry (2016), 55 (16), 7829-7831CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
      α-Factor-1 (WHWLQLKPGQPMY), a peptidic pheromone of Saccharomyces cerevisiae yeast, contains a XHX type copper(II) binding N-terminal site. Using a sol. analog, WHWSKNR-amide, it was shown that the W1H2W3 site alone binds copper(II) with a Kd value of 0.18 pM at pH 7.4 and also binds imidazole (Im) in a ternary complex (Kd of 1 mM at pH 7.4). This interaction boosts the ability of the peptide to sequester copper(II) depending on the Im concn. up to a subfemtomolar range, not available for any oligopeptidic system studied before. Therefore, α-factor-1 and other XHX-type peptides are likely copper(II) carriers in biol. systems.
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      Shahzad, R.; Jones, M. R.; Viles, J. H.; Jones, C. E. Endocytosis of the tachykinin neuropeptide, neurokinin B, in astrocytes and its role in cellular copper uptake. J. Inorg. Biochem. 2016, 162, 319– 325,  DOI: 10.1016/j.jinorgbio.2016.02.027
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      Endocytosis of the tachykinin neuropeptide, neurokinin B, in astrocytes and its role in cellular copper uptake
      Shahzad, Reeha; Jones, Mark R.; Viles, John H.; Jones, Christopher E.
      Journal of Inorganic Biochemistry (2016), 162 (), 319-325CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)
      The tachykinin neuropeptide, neurokinin B (NKB), belongs to a family of peptides having diverse roles in the brain. NKB, along with several other tachykinins, has been identified as a copper-binding peptide, however the physiol. relevance of the binding is unclear. Previously, NKB was shown to limit the ability of copper to enter astrocytes and disrupt calcium homeostasis and it was thought that the peptide was sequestering the metal extracellularly. Here we use a fluorescein-labeled NKB peptide (F-NKB) to show that NKB is not retained extracellularly, but is endocytosed within 10-20 min after addn. to the cell media. The endocytosis is not inhibited when NKB is delivered as a copper-complex, [CuII(F-NKB)2]. Endocytosis of NKB can increase intracellular copper. Comparison to cells cultured in copper-free buffer indicated that apo-NKB can facilitate uptake of copper found in normal culture media. To achieve this NKB must compete with a variety of copper proteins, and we show that NKB can successfully compete with copper-binding peptides derived from the prion protein, itself assocd. with Cu(II) and Zn(II) metab. We suggest a mechanism of receptor mediated endocytosis to account for the observations.
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      Mital, M.; Wezynfeld, N. E.; Frączyk, T.; Wiloch, M. Z.; Wawrzyniak, U. E.; Bonna, A.; Tumpach, C.; Barnham, K. J.; Haigh, C. L.; Bal, W.; Drew, S. C. A Functional Role for Aβ in Metal Homeostasis? N-Truncation and High-Affinity Copper Binding. Angew. Chem., Int. Ed. 2015, 54, 10460– 10464,  DOI: 10.1002/anie.201502644
      6
      A Functional Role for Aβ in Metal Homeostasis? N-Truncation and High-Affinity Copper Binding
      Mital, Mariusz; Wezynfeld, Nina E.; Fraczyk, Tomasz; Wiloch, Magdalena Z.; Wawrzyniak, Urszula E.; Bonna, Arkadiusz; Tumpach, Carolin; Barnham, Kevin J.; Haigh, Cathryn L.; Bal, Wojciech; Drew, Simon C.
      Angewandte Chemie, International Edition (2015), 54 (36), 10460-10464CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Accumulation of the β-amyloid (Aβ) peptide in extracellular senile plaques rich in copper and zinc is a defining pathol. feature of Alzheimer's disease (AD). The Aβ1-x (x=16/28/40/42) peptides have been the primary focus of CuII binding studies for more than 15 years; however, the N-truncated Aβ4-42 peptide is a major Aβ isoform detected in both healthy and diseased brains, and it contains a novel N-terminal FRH sequence. Proteins with His at the third position are known to bind CuII avidly, with conditional log K values at pH 7.4 in the range of 11.0-14.6, which is much higher than that detd. for Aβ1-x peptides. By using Aβ4-16 as a model, it was demonstrated that its FRH sequence stoichiometrically binds CuII with a conditional Kd value of 3×10-14 M at pH 7.4, and that both Aβ4-16 and Aβ4-42 possess negligible redox activity. Combined with the predominance of Aβ4-42 in the brain, our results suggest a physiol. role for this isoform in metal homeostasis within the central nervous system.
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    7. 7
      Stefaniak, E.; Bal, W. CuII Binding Properties of N-Truncated Aβ Peptides: In Search of Biological Function. Inorg. Chem. 2019, 58, 13561– 13577,  DOI: 10.1021/acs.inorgchem.9b01399
      7
      CuII binding properties of N-truncated Aβ peptides: In search of biological function
      Stefaniak, Ewelina; Bal, Wojciech
      Inorganic Chemistry (2019), 58 (20), 13561-13577CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
      A review. As life expectancy increases, the no. of people affected by progressive and irreversible dementia, Alzheimer's Disease (AD), is predicted to grow. No drug designs seem to be working in humans, apparently because the origins of AD have not been identified. Invoking amyloid cascade, metal ions, and ROS prodn. hypothesis of AD, herein we share our point of view on Cu(II) binding properties of Aβ4-x, the most prevalent N-truncated Aβ peptide, currently known as the main constituent of amyloid plaques. The capability of Aβ4-x to rapidly take over copper from previously tested Aβ1-x peptides and form highly stable complexes, redox unreactive and resistant to copper exchange reactions, prompted us to propose physiol. roles for these peptides. We discuss the new findings on the reactivity of Cu(II)Aβ4-x with coexisting biomols. in the context of synaptic cleft; we suggest that the role of Aβ4-x peptides is to quench Cu(II) toxicity in the brain and maintain neurotransmission.
      >> More from SciFinder ®
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    8. 8
      Bal, W.; Jeżowska-Bojczuk, M.; Kasprzak, K. S. Binding of Nickel(II) and Copper(II) to the N-Terminal Sequence of Human Protamine HP2. Chem. Res. Toxicol. 1997, 10, 906– 914,  DOI: 10.1021/tx970028x
      8
      Binding of Nickel(II) and Copper(II) to the N-Terminal Sequence of Human Protamine HP2
      Bal, Wojciech; Jezowska-Bojczuk, Ma-lgorzata; Kasprzak, Kazimierz S.
      Chemical Research in Toxicology (1997), 10 (8), 906-914CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
      A potentiometric and spectroscopic (UV/vis and CD) study of Cu(II) and Ni(II) binding to the N-terminal pentadecapeptide of human protamine HP2 (HP21-15) was performed. The results indicate that the N-terminal tripeptide motif Arg-Thr-His is the exclusive binding site for both metal ions at a metal to HP21-15 molar ratio not higher than 1. The very high value of protonation-cor. stability const. (log *K) for Ni(II)-HP21-15 complex, -19.29, indicates that HP2 has the potential to sequester Ni(II) from other peptide and protein carriers, including albumin. The same is likely for Cu(II) (log *K = -13.13). The CD spectra of Cu(II) and Ni(II) complexes of HP21-15 indicate that the N-terminal metal binding affects the overall conformation of the peptide that, in turn, may alter interaction of HP2 with DNA. These results imply HP2 as a likely target for the toxic metals Ni(II) and Cu(II).
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    9. 9
      Bal, W.; Lukszo, J.; Kasprzak, K. S. Mediation of Oxidative DNA Damage by Nickel(II) and Copper(II) Complexes with the N-Terminal Sequence of Human Protamine HP2. Chem. Res. Toxicol. 1997, 10, 915– 921,  DOI: 10.1021/tx970029p
      9
      Mediation of Oxidative DNA Damage by Nickel(II) and Copper(II) Complexes with the N-Terminal Sequence of Human Protamine HP2
      Bal, Wojciech; Lukszo, Jan; Kasprzak, Kazimierz S.
      Chemical Research in Toxicology (1997), 10 (8), 915-921CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
      The potential of Ni(II) and Cu(II) complexes with Arg-Thr-His-Gly-Gln-Ser-His-Tyr-Arg-Arg-Arg-His-Cys-Ser-Arg-amide (HP21-15), a peptide modeling the N-terminal amino acid sequence of human protamine HP2, to mediate oxidative DNA damage was studied by measurements of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) generation from 2'-deoxyguanosine (dG) and calf thymus DNA and by formation of double-strand breaks in calf thymus DNA. The concns. of reagents were 0.1 mM dG and the metal-HP21-15 complex, 1 mM H2O2, 1.5 mM DNA (per phosphate group), 100 mM phosphate buffer, pH 7.4, ambient O2. Samples were incubated at 37° for 16-24 h. The Cu(II)-HP21-15 complex was found to be an effective promoter of the formation of 8-oxo-dG from both dG and DNA with ambient O2 (approx. 13- and 3-fold increase vs. the oxidant alone, resp.) and H2O2 (approx. 25-fold increase in either case). The Ni(II)-HP21-15 complex was ineffective with O2 vs. 8-oxo-dG prodn. from both substrates but markedly enhanced the attack of H2O2 on dG and DNA (approx. 5-fold increase of 8-oxo-dG prodn. in either case). Both Cu(II)- and Ni(II)-HP21-15 equally promoted double-strand scission by H2O2 in calf thymus DNA. The promotion by the complexes of dG and DNA oxidn. with H2O2 was accompanied by oxidative damage to the complexes themselves, consisting of decreasing contents of their His (to approx. 50% of control in either complex) and esp. Tyr (down to 48% of control in Cu(II)- and 19% in Ni(II)-HP21-15) residues, as well as appearance of aspartic acid, the known oxidn. product of His residues in peptides (up to 22% vs. Gly for Cu(II)- and 10% for Ni(II)-HP21-15). The above results provide a novel chem. mechanism of Cu(II) and Ni(II) toxicity and may have wide implications for reproductive and transgenerational effects of metal exposure.
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    10. 10
      Liang, R.; Senturker, S.; Shi, X.; Bal, W.; Dizdaroglu, M.; Kasprzak, K. S. Effects of Ni(II) and Cu(II) on DNA interaction with the N-terminal sequence of human protamine P2: Enhancement of binding and mediation of oxidative DNA strand scission and base damage. Carcinogenesis 1999, 20, 893– 898,  DOI: 10.1093/carcin/20.5.893
      10
      Effects of Ni(II) and Cu(II) on DNA interaction with the N-terminal sequence of human protamine P2: enhancement of binding and mediation of oxidative DNA strand scission and base damage
      Liang, Rongti; Senturker, Sema; Shi, Xianglin; Bal, Wojciech; Dizdaroglu, Miral; Kasprzak, Kazimierz S.
      Carcinogenesis (1999), 20 (5), 893-898CODEN: CRNGDP; ISSN:0143-3334. (Oxford University Press)
      Epidemiol. evidence suggests that certain paternal exposures to metals may increase the risk of cancer in the progeny. This effect may be assocd. with promutagenic damage to the sperm DNA. The latter is packed with protamines which might sequester carcinogenic metals and moderate the damage. Human protamine P2 has an amino acid motif at its N-terminus that can serve as a heavy metal trap, esp. for Ni(II) and Cu(II). We have synthesized a pentadecapeptide modeling this motif, Arg-Thr-His-Gly-Gln-Ser-His-Tyr-Arg-Arg-Arg-His-Cys-Ser-Arg-amide (HP21-15) and described its complexes with Ni(II) and Cu(II), including their capacity to mediate oxidative DNA degrdn. (1997). In the present study, effects of HP21-15 on Ni(II)- and Cu(II)-mediated DNA oxidn. by H2O2 at pH 7.4 were investigated in more detail using the circular plasmid pUC19 DNA as a target, and the single/double-strand breaks and prodn. of oxidized DNA bases, as end points. Ni(II) alone was found to promote oxidative DNA strand scission (mostly single strand breaks) and base damage, while Cu(II) alone produced the same effects, but to a much greater extent. Both metals were relatively more damaging to the pyrimidine bases than to purine bases. HP21-15 tended to increase the Ni(II)/H2O2-induced DNA breakage. In sharp contrast, the destruction of DNA strands by Cu(II)/H2O2 was almost completely prevented by HP21-15. The effect of HP21-15 on the oxidative DNA base damage varied from a limited enhancement (5-hydroxyhydantoin and thymine glycol) to slight suppression (5-hydroxycytosine, 5-hydroxyuracil, 8-oxoguanine, 8-oxoadenine, 2-hydroxyadenine, fapyguanine and fapyadenine) toward Ni(II)/H2O2. HP21-15 strongly suppressed the oxidative activity of Cu(II)/H2O2 in regard to all bases in DNA. Consistently with the above, the ESR/spin trap measurements revealed greater and more persistent generation of OH· and O2-•-like oxidants from H2O2 by the Ni(II)-HP21-15 complex than by the Cu(II)-HP21-15 complex (no O2-•-was detected). Both complexes were also found to bind to DNA more strongly than HP21-15 alone. The results indicate that protamine P2 is capable of binding Ni(II) and Cu(II) and, in this way, attenuating the mediation of oxidative DNA damage by Cu(II), but not Ni(II). The effects found may be mechanistically involved in the reproductive toxicity and carcinogenicity of metals.
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    11. 11
      Conklin, S. E.; Bridgman, E. C.; Su, Q.; Riggs-Gelasco, P.; Haas, K. L.; Franz, K. J. Specific Histidine Residues Confer Histatin Peptides with Copper-Dependent Activity against Candida albicans. Biochemistry 2017, 56, 4244– 4255,  DOI: 10.1021/acs.biochem.7b00348
      11
      Specific Histidine Residues Confer Histatin Peptides with Copper-Dependent Activity against Candida albicans
      Conklin, Steven E.; Bridgman, Emma C.; Su, Qiang; Riggs-Gelasco, Pamela; Haas, Kathryn L.; Franz, Katherine J.
      Biochemistry (2017), 56 (32), 4244-4255CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The histidine-rich salivary peptides of the histatin family are known to bind copper (Cu) and other metal ions in vitro, but the details of these interactions are poorly understood and their implications on in vivo antifungal activity have not been established. Here, we show that availability of Cu during exposure of Candida albicans to histatin-5 (Hist-5) modulates its antifungal activity. Antifungal susceptibility testing revealed that cotreatment of Hist-5 with Cu improved the EC50 from ∼5 μM to ∼1 μM, whereas cotreatment with a high-affinity Cu-specific chelator abrogated antifungal activity. Spectrophotometric titrns. revealed two previously unrecognized Cu(I) binding sites with apparent Kd values at pH 7.4-20 nM, and confirmed a high-affinity Cu(II) binding site at the Hist-5 N-terminus with apparent Kd ∼ 8 pM. Evaluation of a series of His-to-Ala peptides contg. the first 12 residues of Hist-5 identified adjacent His residues (bis-His) as crit. anchors for Cu(I) binding, and the presence of a third ligand was revealed by X-ray absorption spectroscopy (XAS). On their own, the truncated peptides were ineffective at inhibiting growth of C. albicans, but treatment with supplemental Cu resulted in EC50 values down to ∼5 μM, approaching that of full-length Hist-5. The efficacy of the peptides depended on an intact bis-His site and correlated with Cu(I) affinity. Together, these results establish new structure-function relationships linking specific histidine residues with Cu-binding affinity and antifungal activity, and provide further evidence for the involvement of metals in modulating the biol. activity of these antifungal peptides.
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    12. 12
      Frączyk, T. Cu(II)-Binding N-Terminal Sequences of Human Proteins. Chem. Biodiversity 2021, 18, e2100043  DOI: 10.1002/cbdv.202100043
      12
      Cu(II)-Binding N-Terminal Sequences of Human Proteins
      Fraczyk, Tomasz
      Chemistry & Biodiversity (2021), 18 (4), e2100043CODEN: CBHIAM; ISSN:1612-1872. (Verlag Helvetica Chimica Acta)
      Proteins anchor copper(II) ions mainly by imidazole from histidine residues located in different positions in the primary protein structures. However, the motifs with histidine in the first three N-terminal positions (His1, His2, and His3) show unique Cu(II)-binding properties, such as availability from the surface of the protein, high flexibility, and high Cu(II) exchangeability with other ligands. It makes such sequences beneficial for the fast exchange of Cu(II) between ligands. Furthermore, sequences with His1 and His2, thus, non-satg. the Cu(II) coordination sphere, are redox-active and may play a role in Cu(II) redn. to Cu(I). All human protein sequences deposited in UniProt Knowledgebase were browsed for those contg. His1, His2, or His3. Proteolytically modified sequences (with the removal of a propeptide or Met residue) were taken for the anal. Finally, the sequences were sorted out according to the subcellular localization of the proteins to match the resp. sequences with the probability of interaction with divalent copper.
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    13. 13
      Sokolowska, M.; Krezel, A.; Dyba, M.; Szewczuk, Z.; Bal, W. Short peptides are not reliable models of thermodynamic and kinetic properties of the N-terminal metal binding site in serum albumin. Eur. J. Biochem. 2002, 269, 1323– 1331,  DOI: 10.1046/j.1432-1033.2002.02772.x
      13
      Short peptides are not reliable models of thermodynamic and kinetic properties of the N-terminal metal binding site in serum albumin
      Sokolowska, Magdalena; Krezel, Artur; Dyba, Marcin; Szewczuk, Zbigniew; Bal, Wojciech
      European Journal of Biochemistry (2002), 269 (4), 1323-1331CODEN: EJBCAI; ISSN:0014-2956. (Blackwell Publishing Ltd.)
      A comparative study of thermodn. and kinetic aspects of Cu(II) and Ni(II) binding at the N-terminal binding site of human and bovine serum albumins (HSA and BSA, resp.) and short peptide analogs was performed using potentiometry and spectroscopic techniques. It was found that while qual. aspects of interaction (spectra and structures of complexes, order of reactions) could be reproduced, the quant. parameters (stability and rate consts.) could not. The N-terminal site in HSA is much more similar to BSA than to short peptides reproducing the HSA sequence. A very strong influence of phosphate ions on the kinetics of Ni(II) interaction was found. This study demonstrates the limitations of short peptide modeling of Cu(II) and Ni(II) transport by albumins.
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    14. 14
      Haas, K. L.; Putterman, A. B.; White, D. R.; Thiele, D. J.; Franz, K. J. Model peptides provide new insights into the role of histidine residues as potential ligands in human cellular copper acquisition via Ctr1. J. Am. Chem. Soc. 2011, 133, 4427– 4437,  DOI: 10.1021/ja108890c
      14
      Model Peptides Provide New Insights into the Role of Histidine Residues as Potential Ligands in Human Cellular Copper Acquisition via Ctr1
      Haas, Kathryn L.; Putterman, Allison B.; White, Daniel R.; Thiele, Dennis J.; Franz, Katherine J.
      Journal of the American Chemical Society (2011), 133 (12), 4427-4437CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Cellular acquisition of copper in eukaryotes is primarily accomplished through the Ctr family of copper transport proteins. In both humans and yeast, methionine-rich "Mets" motifs in the amino-terminal extracellular domain of Ctr1 are thought to be responsible for recruitment of copper at the cell surface. Unlike yeast, mammalian Ctr1 also contains extracellular histidine-rich motifs, although a role for these regions in copper uptake has not been explored in detail. Herein, synthetic model peptides contg. the first 14 residues of the extracellular domain of human Ctr1 (MDHSHHMGMSYMDS) have been prepd. and evaluated for their apparent binding affinity to both Cu(I) and Cu(II). These studies reveal a high affinity Cu(II) binding site (log K = 11.0 ± 0.3 at pH 7.4) at the amino-terminus of the peptide as well as a high affinity Cu(I) site (log K = 10.2 ± 0.2 at pH 7.4) that utilizes adjacent HH residues along with an addnl. His or Met ligand. These model studies suggest that the histidine domains may play a direct role in copper acquisition from serum copper-binding proteins and in facilitating the redn. of Cu(II) to the active Ctr1 substrate, Cu(I). We tested this hypothesis by expressing a Ctr1 mutant lacking only extracellular histidine residues in Ctr1-knockout mouse embryonic fibroblasts. Results from live cell studies support the hypothesis that extracellular amino-terminal His residues directly participate in the copper transport function of Ctr1.
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    15. 15
      Stefaniak, E.; Płonka, D.; Drew, S. C.; Bossak-Ahmad, K.; Haas, K. L.; Pushie, M. J.; Faller, P.; Wezynfeld, N. E.; Bal, W. The N-terminal 14-mer model peptide of human Ctr1 can collect Cu(ii) from albumin. Implications for copper uptake by Ctr1. Metallomics 2018, 10, 1723– 1727,  DOI: 10.1039/C8MT00274F
      15
      The N-terminal 14-mer model peptide of human Ctr1 can collect Cu(II) from albumin. Implications for copper uptake by Ctr1
      Stefaniak, Ewelina; Plonka, Dawid; Drew, Simon C.; Bossak-Ahmad, Karolina; Haas, Kathryn L.; Pushie, M. Jake; Faller, Peter; Wezynfeld, Nina E.; Bal, Wojciech
      Metallomics (2018), 10 (12), 1723-1727CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)
      Human cells acquire copper primarily via the copper transporter 1 protein, hCtr1. We demonstrate that at extracellular pH 7.4 CuII is bound to the model peptide hCtr11-14via an ATCUN motif and such complexes are strong enough to collect CuII from albumin, supporting the potential physiol. role of CuII binding to hCtr1.
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    16. 16
      Arena, G.; La Mendola, D.; Pappalardo, G.; Sóvágó, I.; Rizzarelli, E. Interactions of Cu2+ with prion family peptide fragments: Considerations on affinity, speciation and coordination. Coord. Chem. Rev. 2012, 256, 2202– 2218,  DOI: 10.1016/j.ccr.2012.03.038
      16
      Interactions of Cu2+ with prion family peptide fragments: Considerations on affinity, speciation and coordination
      Arena, Giuseppe; La Mendola, Diego; Pappalardo, Giuseppe; Sovago, Imre; Rizzarelli, Enrico
      Coordination Chemistry Reviews (2012), 256 (19-20), 2202-2218CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)
      A review. The review describes the stability and the coordination modes of Cu2+ complexes with different regions of N-terminus prion proteins. The structural features of the different metal species are correlated both with the Cu2+-driven redox properties and with the conformational changes induced by the Cu2+ in the different metal binding regions of the protein. The formation of mixed metal complexes is also discussed. We emphasize that binding features should be discussed by referring to the species that actually forms under specific conditions (pH, buffer, etc.) rather than to the 'binding site'; correlating properties with the structures of the so called 'binding sites' may lead to misinterpretation of the exptl. results, since a 'binding site' often corresponds to a mixt. of species. We also highlight that ignoring species that form with ligands other than the prion peptide (e.g. the buffer) may lead to underestimating their role in crucial processes (e.g. redox activity).
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    17. 17
      Zawisza, I.; Rózga, M.; Bal, W. Affinity of copper and zinc ions to proteins and peptides related to neurodegenerative conditions (Aβ, APP, α-synuclein, PrP). Coord. Chem. Rev. 2012, 256, 2297– 2307,  DOI: 10.1016/j.ccr.2012.03.012
      17
      Affinity of copper and zinc ions to proteins and peptides related to neurodegenerative conditions (Aβ, APP, α-synuclein, PrP)
      Zawisza, Izabela; Rozga, Malgorzata; Bal, Wojciech
      Coordination Chemistry Reviews (2012), 256 (19-20), 2297-2307CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)
      A review. The review describes the state of the art in the field of stability const. detn. for Cu(II), Cu(I) and Zn(II) complexes of proteins and peptides involved in neurodegenerative diseases, α-synuclein (aS), prion protein (PrP), amyloid precursor protein (APP) and amyloid β peptides (Aβ). The methodologies and results are critically analyzed and recommendations are formulated about possible systematic errors in these studies. The possibility of formation of ternary complexes with titrn. competitors is discussed.
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    18. 18
      Gonzalez, P.; Bossak, K.; Stefaniak, E.; Hureau, C.; Raibaut, L.; Bal, W.; Faller, P. N-Terminal Cu-Binding Motifs (Xxx-Zzz-His, Xxx-His) and Their Derivatives: Chemistry, Biology and Medicinal Applications. Chem. - Eur. J. 2018, 24, 8029– 8041,  DOI: 10.1002/chem.201705398
      18
      N-terminal Cu-binding motifs (Xxx-Zzz-His, Xxx-His) and their derivatives: Chemistry, biology and medicinal applications
      Gonzalez, Paulina; Bossak, Karolina; Stefaniak, Ewelina; Hureau, Christelle; Raibaut, Laurent; Bal, Wojciech; Faller, Peter
      Chemistry - A European Journal (2018), 24 (32), 8029-8041CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Peptides and proteins with N-terminal amino acid sequences NH2-Xxx-His (XH) and NH2-Xxx-Zzz-His (XZH) form well-established high-affinity CuII-complexes. Key examples are Asp-Ala-His (in serum albumin) and Gly-His-Lys, the wound-healing factor. This opens a straightforward way to add a high-affinity CuII-binding site to almost any peptide or protein, by chem. or recombinant approaches. Thus, these motifs, NH2-Xxx-Zzz-His in particular, have been used to equip peptides and proteins with a multitude of functions based on the redox activity of Cu, including nuclease, protease, glycosidase, or oxygen activation properties, useful in anticancer or antimicrobial drugs. More recent research suggests novel biol. functions, mainly based on the redox inertness of CuII in XZH, like PET imaging (with 64Cu), chelation therapies (for instance in Alzheimer's disease and other types of neurodegeneration), antioxidant units, Cu transporters, and activation of biol. functions by strong CuII binding. Here, the authors provide an overview of the chem. properties of Cu-XH and -XZH motifs and discuss the pros and cons of the vastly different biol. applications, and how they could be improved depending on the application.
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    19. 19
      Nagaj, J.; Stokowa-Sołtys, K.; Zawisza, I.; Jeżowska-Bojczuk, M.; Bonna, A.; Bal, W. Selective control of Cu(II) complex stability in histidine peptides by β-alanine. J. Inorg. Biochem. 2013, 119, 85– 89,  DOI: 10.1016/j.jinorgbio.2012.11.002
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      Selective control of Cu(II) complex stability in histidine peptides by β-alanine
      Nagaj, Justyna; Stokowa-Soltys, Kamila; Zawisza, Izabela; Jezowska-Bojczuk, Malgorzata; Bonna, Arkadiusz; Bal, Wojciech
      Journal of Inorganic Biochemistry (2013), 119 (), 85-89CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)
      The cooperativity of formation of 5-membered and 6-membered chelate rings is the driving force for specificity and selectivity in Cu(II) peptidic complexes. α-Amino acids enable the formation of 5-membered rings, while a 6-membered ring is provided by the coordination of the His side chain imidazole. Introduction of β-alanine is another way of creating a 6-membered ring in the Cu(II) complex. The potentiometric and spectroscopic (UV-vis and CD) study of Cu(II) complexation by a series of four peptides, AAH-am, ABH-am, BAH-am, and BBH-am (where B stands for β-alanine, and -am for C-terminal amide) revealed a very strong effect of the sizes of individual rings, with the order of complex stability AAH-am (5,5,6) > BAH-am (6,5,6) > ABH-am (5,6,6) » BBH-am (6,6,6). The stabilities of ABH-am and BAH-am complexes are intermediate between those of strong His-3 peptides but these complexes are still able to sat. the coordination sphere of the Cu(II) ion at neutral pH. This fact opens up new possibilities in engineering specific peptide-based chelates.
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    20. 20
      Kotuniak, R.; Strampraad, M. J. F.; Bossak-Ahmad, K.; Wawrzyniak, U. E.; Ufnalska, I.; Hagedoorn, P.-L.; Bal, W. Key Intermediate Species Reveal the Copper(II)-Exchange Pathway in Biorelevant ATCUN/NTS Complexes. Angew. Chem., Int. Ed. 2020, 59, 11234– 11239,  DOI: 10.1002/anie.202004264
      20
      Key Intermediate Species Reveal the Copper(II)-Exchange Pathway in Biorelevant ATCUN/NTS Complexes
      Kotuniak, Radoslaw; Strampraad, Marc J. F.; Bossak-Ahmad, Karolina; Wawrzyniak, Urszula E.; Ufnalska, Iwona; Hagedoorn, Peter-Leon; Bal, Wojciech
      Angewandte Chemie, International Edition (2020), 59 (28), 11234-11239CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The amino-terminal copper and nickel/N-terminal site (ATCUN/NTS) present in proteins and bioactive peptides exhibits high affinity towards CuII ions and have been implicated in human copper physiol. Little is known, however, about the rate and exact mechanism of formation of such complexes. We used the stopped-flow and microsecond freeze-hyperquenching (MHQ) techniques supported by steady-state spectroscopic and electrochem. data to demonstrate the formation of partially coordinated intermediate CuII complexes formed by glycyl-glycyl-histidine (GGH) peptide, the simplest ATCUN/NTS model. One of these novel intermediates, characterized by two-nitrogen coordination, t1/2≈100 ms at pH 6.0 and the ability to maintain the CuII/CuI redox pair is the best candidate for the long-sought reactive species in extracellular copper transport.
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    21. 21
      Bossak-Ahmad, K.; Frączyk, T.; Bal, W.; Drew, S. C. The Sub-picomolar Cu2+ Dissociation Constant of Human Serum Albumin. ChemBioChem 2020, 21, 331– 334,  DOI: 10.1002/cbic.201900435
      21
      The Sub-picomolar Cu2+ Dissociation Constant of Human Serum Albumin
      Bossak-Ahmad, Karolina; Fraczyk, Tomasz; Bal, Wojciech; Drew, Simon C.
      ChemBioChem (2020), 21 (3), 331-334CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The apparent affinity of human serum albumin (HSA) for divalent copper has long been the subject of great interest, due to its presumed role as the major Cu2+-binding ligand in blood and cerebrospinal fluid. Using a combination of electronic absorption, CD and room-temp. ESR spectroscopies, together with potentiometric titrns., we competed the tripeptide GGH against HSA to reveal a conditional binding const. of log cKCuCu(HSA)=13.02±0.05 at pH 7.4. This rigorously detd. value of the Cu2+ affinity has important implications for understanding the extracellular distribution of copper.
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    22. 22
      Magrì, A.; Tabbì, G.; Giuffrida, A.; Pappalardo, G.; Satriano, C.; Naletova, I.; Nicoletti, V. G.; Attanasio, F. Influence of the N-terminus acetylation of Semax, a synthetic analog of ACTH(4–10), on copper(II) and zinc(II) coordination and biological properties. J. Inorg. Biochem. 2016, 164, 59– 69,  DOI: 10.1016/j.jinorgbio.2016.08.013
      22
      Influence of the N-terminus acetylation of Semax, a synthetic analog of ACTH(4-10), on copper(II) and zinc(II) coordination and biological properties
      Magri, Antonio; Tabbi, Giovanni; Giuffrida, Alessandro; Pappalardo, Giuseppe; Satriano, Cristina; Naletova, Irina; Nicoletti, Vincenzo G.; Attanasio, Francesco
      Journal of Inorganic Biochemistry (2016), 164 (), 59-69CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)
      Semax is a heptapeptide (Met-Glu-His-Phe-Pro-Gly-Pro) that encompasses the sequence 4-7 of N-terminal domain of the adrenocorticotropic hormone and a C-terminal Pro-Gly-Pro tripeptide. N-terminal amino group acetylation (Ac-Semax) modulates the chem. and biol. properties of parental peptide, modifying the ability of Semax to form complex species with Cu(II) ion. At physiol. pH, the main complex species formed by Ac-Semax, [CuLH-2]2-, consists in a distorted CuN3O chromophore with a weak apical interaction of the methionine sulfur. Such a complex differs from the Cu(II)-Semax complex system, which exhibits a CuN4 chromophore. The reduced ligand field affects the [CuLH-2]2- formal redox potential, which is more pos. than that of Cu(II)-Semax corresponding species. In the amino-free form, the resulting complex species is redox-stable and unreactive against ascorbic acid, unlike the acetylated form. Semax acetylation did not protect from Cu(II) induced toxicity on a SH-SY5Y neuroblastoma cell line, thus demonstrating the crucial role played by the free NH2 terminus in the cell protection. Since several brain diseases are assocd. either to Cu(II) or Zn(II) dyshomeostasis, here we characterized also the complex species formed by Zn(II) with Semax and Ac-Semax. Both peptides were able to form Zn(II) complex species with comparable strength. Confocal microscopy imaging confirmed that peptide group acetylation does not affect the Zn(II) influx in neuroblastoma cells. Moreover, a punctuate distribution of Zn(II) within the cells suggests a preferred subcellular localization that might explain the zinc toxic effect. A future perspective can be the use of Ac-Semax as ionophore in antibody drug conjugates to produce a dysmetallostasis in tumor cells.
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    23. 23
      Gavriilidou, A. F. M.; Gülbakan, B.; Zenobi, R. Influence of Ammonium Acetate Concentration on Receptor-Ligand Binding Affinities Measured by Native Nano ESI-MS: A Systematic Study. Anal. Chem. 2015, 87, 10378– 10384,  DOI: 10.1021/acs.analchem.5b02478
      23
      Influence of Ammonium Acetate Concentration on Receptor-Ligand Binding Affinities Measured by Native Nano ESI-MS: A Systematic Study
      Gavriilidou, Agni F. M.; Gulbakan, Basri; Zenobi, Renato
      Analytical Chemistry (Washington, DC, United States) (2015), 87 (20), 10378-10384CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Native electrospray ionization (ESI) mass spectrometry (MS) is a powerful technique for analyzing biomols. in their native state. However, ESI-MS is incompatible with nonvolatile soln. additives. Therefore, biomols. have to be electrosprayed from a soln. that differs from their purifn. or storage buffer, often aq. ammonium acetate (AmAc). In this study, the effect of the ionic strength on the dissocn. consts. of six different noncovalent complexes, that cover interactions present in many biol. systems, was investigated. Complexes were electrosprayed from 10 mM, 50 mM, 100 mM, 300 mM, and 500 mM aq. AmAc. For all systems, it was shown that the binding affinity is significantly influenced by the ionic strength of the soln. The detd. dissocn. const. (Kd) was affected more than 50% when increasing the AmAc concn. The results are interpreted in terms of altered ionic interactions induced by the soln. This work emphasizes the modulating effect of the ions on noncovalent interactions and the importance of carefully choosing the AmAc concn. for quantifying the receptor-ligand binding strengths.
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    24. 24
      Erba, E. B.; Zenobi, R. Mass spectrometric studies of dissociation constants of noncovalent complexes. Annu. Rep. Prog. Chem., Sect. C: Phys. Chem. 2011, 107, 199,  DOI: 10.1039/c1pc90006d
      24
      Mass spectrometric studies of dissociation constants of noncovalent complexes
      Erba, Elisabetta Boeri; Zenobi, Renato
      Annual Reports on the Progress of Chemistry, Section C: Physical Chemistry (2011), 107 (), 199-228CODEN: ACPCDW; ISSN:0260-1826. (Royal Society of Chemistry)
      A review. Specific interactions among biomols. to form noncovalently bound complexes play a pivotal role in key cellular processes such as cell division, cell signalling, gene transcription and translation. The propensity of noncovalently bound complexes to dissoc. into their components can be quantified and consts. of dissocn. (Kd) can be obtained by various methods. Mass spectrometry has become an important method to measure Kd. The advent of soft ionisation techniques, in particular electrospray ionisation (ESI) and matrix-assisted laser desorption/ionisation (MALDI) has established mass spectrometry as a viable technique for investigating noncovalent interactions and for quantifying their binding strengths. Under carefully chosen exptl. and instrumental conditions, it is possible to observe intact noncovalent complexes in the gas phase using ESI and MALDI, and to use the mass spectra as a read-out for detg. soln.-phase Kd. Compared to other biophys. methods, mass spectrometry is highly sensitive and fast, and gives addnl. information about the stoichiometry and specificity of noncovalent interactions. This review focuses on recent MS-based methodologies for quantification of binding strengths, in particular those that promise to complement conventional biophys. methods.
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    25. 25
      Daniel, J. M.; McCombie, G.; Wendt, S.; Zenobi, R. Mass spectrometric determination of association constants of adenylate kinase with two noncovalent inhibitors. J. Am. Soc. Mass Spectrom. 2003, 14, 442– 448,  DOI: 10.1016/S1044-0305(03)00132-6
      25
      Mass spectrometric determination of association constants of adenylate kinase with two noncovalent inhibitors
      Daniel, J. urg M.; McCombie, Gregor; Wendt, Silke; Zenobi, Renato
      Journal of the American Society for Mass Spectrometry (2003), 14 (5), 442-448CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Science Inc.)
      Noncovalent complexes between chicken muscle adenylate kinase and two inhibitors, P1,P4-di(adenosine-5')tetraphosphate (Ap4A) and P1,P5-di(adenosine-5') pentaphosphate (Ap5A), were investigated with electrospray ionization mass spectrometry under non-denaturing conditions. The noncovalent nature and the specificity of the complexes are demonstrated with a no. of control expts. Titrn. expts. allowed the assocn. consts. for inhibitor binding to be detd. Problems with concn. dependent ion yields are circumvented by a data evaluation method that is insensitive to the overall ionization efficiency. The Ka values found were 9.0 × 104 M-1 (Ap4A) and 4.0 × 107 M-1 (Ap5A), resp., in very good agreement with available literature data.
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    26. 26
      Peschke, M.; Verkerk, U. H.; Kebarle, P. Features of the ESI mechanism that affect the observation of multiply charged noncovalent protein complexes and the determination of the association constant by the titration method. J. Am. Soc. Mass Spectrom. 2004, 15, 1424– 1434,  DOI: 10.1016/j.jasms.2004.05.005
      26
      Features of the ESI mechanism that affect the observation of multiply charged noncovalent protein complexes and the determination of the association constant by the titration method
      Peschke, Michael; Verkerk, Udo H.; Kebarle, Paul
      Journal of the American Society for Mass Spectrometry (2004), 15 (10), 1424-1434CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)
      Several factors, attributable to the ESIMS mechanism, that can affect the assumptions of the titrn. method are examd.: (1) The assumption that the concns. in soln. of the protein P, the ligand L, and the complex PL are proportional to the resp. ion intensities obsd. with ESIMS, is examd. with expts. in which ion intensities of two non-interacting proteins are compared with the resp. concns. The intensities are found to be approx. proportional to the concns. The proportionality factors are found to increase as the mass of the protein is decreased. Very small proteins have much higher intensities. The results suggest that it is preferable to use only the intensity ratio of PL and P, whose masses are very close to each other when L is small, to det. the assocn. const. KA in soln. (2) From the charge residue model (CRM) one expects that the soln. will experience a very large increase of concn. due to evapn. of the precursor droplets, before the proteins P and PL are produced in the gas phase. This can shift the equil. in the droplets: P + L = PL, towards PL. Anal. of the droplet evapn. history shows that such a shift is not likely, because the time of droplet evolution is very short, only several μs, and the equil. relaxation time is much longer. (3) The droplet history shows that unreacted P and L can be often present together in the same droplet. On complete evapn. of such droplets L will land on P leading to PL and this effect will lead to values of KA that are too high. However, it is argued that mostly accidental, weakly bonded, complexes will form and these will dissoc. in the clean up stages (heated transfer capillary and CAD region). Thus only very small errors are expected due to this cause. (4) Some PL complexes may have bonding that is too weak in the gas phase even though they have KA values in soln. that predict high soln. PL yields. In this case the PL complexes may decomp. in the clean up stages and not be obsd. with sufficient intensity in the mass spectrum. This will lead to KA values that are too low. The effect is expected for complexes that involve significant hydrophobic interaction that leads to high stability of the complex in soln. but low stability in the gas phase. The titrn. method is not suited for such systems.
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    27. 27
      Zhang, S.; van Pelt, C. K.; Wilson, D. B. Quantitative determination of noncovalent binding interactions using automated nanoelectrospray mass spectrometry. Anal. Chem. 2003, 75, 3010– 3018,  DOI: 10.1021/ac034089d
      27
      Quantitative determination of noncovalent binding interactions using automated nanoelectrospray mass spectrometry
      Zhang, Sheng; Van Pelt, Colleen K.; Wilson, David B.
      Analytical Chemistry (2003), 75 (13), 3010-3018CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Electrospray ionization mass spectrometry (ESI-MS) has proven to be an extremely powerful tool for studying biomol. structures and noncovalent interactions. Here we report a method using a fully automated, chip-based nanoESI-MS system to det. the dissocn. consts. (Kd) for the complexes of two different proteins with their ligands. The automated nanoelectrospray system, consisting of the NanoMate and ESI chip, serves functionally as a combination of autosampler and nanoelectrospray ionization source. This system provides all the advantages of conventional nanoelectrospray plus automated, high-throughput analyses without carryover. The automated nanoESI system was used to investigate quant. noncovalent interactions between RNase A (RNase A) and cytidylic acid ligands (2'-CMP, CTP), a well-characterized model protein-ligand complex, and between an inactive endocellulase mutant (Thermobifida fusca Cel6A D117Acd) and four oligosaccharide ligands (cellotriose, cellotetraose, cellopentaose, cellohexaose). Both titrn. and competitive binding approaches were performed prior to automated nanoESI-MS anal. with a Q-TOF mass spectrometer. Dissocn. consts. for each complex were calcd. from the sum of ion peak areas of free and complexed proteins during the titrn. and competition expts. The measured Kd values for the RNase A-CMP and Cel6A D117Acd-G3 complexes were found to be in excellent agreement with the available published values obtained by std. spectroscopic titrn. techniques. To our knowledge, this is the first report of using an ESI-MS approach to study the interactions between a cellulase and oligosaccharides. The results provide new insights for understanding the nature of cellulase-cellulose interactions.
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    28. 28
      Carlton, D. D., Jr; Schug, K. A. A review on the interrogation of peptide-metal interactions using electrospray ionization-mass spectrometry. Anal. Chim. Acta 2011, 686, 1– 39,  DOI: 10.1016/j.aca.2010.11.050
      There is no corresponding record for this reference.
    29. 29
      Jecklin, M. C.; Touboul, D.; Bovet, C.; Wortmann, A.; Zenobi, R. Which electrospray-based ionization method best reflects protein-ligand interactions found in solution? a comparison of ESI, nanoESI, and ESSI for the determination of dissociation constants with mass spectrometry. J. Am. Soc. Mass Spectrom. 2008, 19, 332– 343,  DOI: 10.1016/j.jasms.2007.11.007
      29
      Which electrospray-based ionization method best reflects protein-ligand interactions found in solution? A comparison of ESI, nanoESI, and ESSI for the determination of dissociation constants with mass spectrometry
      Jecklin, Matthias Conradin; Touboul, David; Bovet, Cedric; Wortmann, Arno; Zenobi, Renato
      Journal of the American Society for Mass Spectrometry (2008), 19 (3), 332-343CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)
      The authors present a comparison of three different electrospray-based ionization techniques for the investigation of noncovalent complexes with mass spectrometry. The features and characteristics of std. electrospray ionization (ESI), chip-based nanoESI, and electrosonic spray ionization (ESSI) mounted onto a hybrid quadrupole time-of-flight mass spectrometer were compared in their performance to det. the dissocn. const. (KD) of the model system hen egg white lysozyme (HEWL) binding to N,N',N''-triacetylchitotriose (NAG3). The best KD value compared with soln. data were found for ESSI, 19.4 ± 3.6 μM. Then, the authors detd. the KDs of the two nucleotide binding sites of adenylate kinase (AK), where the authors obtained KDs of 2.2 ± 0.8 μM for the first and 19.5 ± 8.0 μM for the second binding site using ESSI. The authors found a weak charge state dependence of the KD for both protein-ligand systems, where for all ionization techniques the KD value decreases with increasing charge state. The authors demonstrate that ESSI is very gentle and insensitive to instrumental parameters, and the KD obtained is in good agreement with soln. phase results from the literature. In addn., the authors tried to det. the KD for the lymphocyte-specific kinase LCK binding to a kinase inhibitor using nanoESI due to the very low amt. of sample available. In this case, the authors found KD values with a strong charge state dependence, which were in no case close to literature values for soln. phase.
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    30. 30
      Kitova, E. N.; El-Hawiet, A.; Schnier, P. D.; Klassen, J. S. Reliable Determinations of Protein-Ligand Interactions by Direct ESI-MS Measurements. Are We There Yet?. J. Am. Soc. Mass Spectrom. 2012, 23, 431– 441,  DOI: 10.1007/s13361-011-0311-9
      30
      Reliable determinations of protein-ligand interactions by direct ESI-MS measurements. Are we there yet?
      Kitova, Elena N.; El-Hawiet, Amr; Schnier, Paul D.; Klassen, John S.
      Journal of the American Society for Mass Spectrometry (2012), 23 (3), 431-441CODEN: JAMSEF; ISSN:1044-0305. (Springer)
      A review. The assocn.-dissocn. of noncovalent interactions between protein and ligands, such as other proteins, carbohydrates, lipids, DNA, or small mols., are crit. events in many biol. processes. The discovery and characterization of these interactions is essential to a complete understanding of biochem. reactions and pathways and to the design of novel therapeutic agents that may be used to treat a variety of diseases and infections. Over the last 20 y, electrospray ionization mass spectrometry (ESI-MS) has emerged as a versatile tool for the identification and quantification of protein-ligand interactions in vitro. Here, the authors describe the implementation of the direct ESI-MS assay for the detn. of protein-ligand binding stoichiometry and affinity. Addnl., the authors outline common sources of error encountered with these measurements and various strategies to overcome them. Finally, the authors comment on some of the outstanding challenges assocd. with the implementation of the assay and highlight new areas where direct ESI-MS measurements are expected to make significant contributions in the future.
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    31. 31
      Smirnova, J.; Zhukova, L.; Witkiewicz-Kucharczyk, A.; Kopera, E.; Olędzki, J.; Wysłouch-Cieszyńska, A.; Palumaa, P.; Hartwig, A.; Bal, W. Quantitative electrospray ionization mass spectrometry of zinc finger oxidation: The reaction of XPA zinc finger with H2O2. Anal. Biochem. 2007, 369, 226– 231,  DOI: 10.1016/j.ab.2007.05.019
      31
      Quantitative electrospray ionization mass spectrometry of zinc finger oxidation: The reaction of XPA zinc finger with H2O2
      Smirnova, Julia; Zhukova, Liliya; Witkiewicz-Kucharczyk, Aleksandra; Kopera, Edyta; Oledzki, Jacek; Wyslouch-Cieszynska, Aleksandra; Palumaa, Peep; Hartwig, Andrea; Bal, Wojciech
      Analytical Biochemistry (2007), 369 (2), 226-231CODEN: ANBCA2; ISSN:0003-2697. (Elsevier)
      Oxidn. plays an important role in the functioning of zinc fingers (ZFs). Electrospray ionization mass spectrometry (ESI-MS) is a very useful technique to study products of ZF oxidn., but its application has been limited largely to qual. anal. of reaction products. ESI-MS has been applied successfully on several occasions to det. binding consts. in metalloproteins. The authors used a synthetic 37-residue peptide acetyl-DYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf), which corresponds to the Cys 4 ZF sequence of human nucleotide excision repair protein XPA, to find out whether ESI-MS might be used quant. to study ZF reaction kinetics. For this purpose, the authors studied oxidn. of the Zn(II) complex of XPAzf (ZnXPAzf) by H2O2 using three techniques in parallel: HPLC of covalent reaction products, 4-(2-pyridylazo)-resorcinol monosodium salt (PAR)-based spectrophotometric zinc release assay, and ESI-MS. Single and double intrapeptide disulfides were detected by ESI-MS to be the sole reaction products. All three techniques yielded independently the same reaction rate, thereby demonstrating that ESI-MS may indeed be used in quant. kinetic studies of ZF reactions. The comparison of exptl. information demonstrated that the formation of the Cys 5Cys 8 single disulfide was responsible for zinc release.
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    32. 32
      Shoshan, M. S.; Dekel, N.; Goch, W.; Shalev, D. E.; Danieli, T.; Lebendiker, M.; Bal, W.; Tshuva, E. Y. Unbound position II in MXCXXC metallochaperone model peptides impacts metal binding mode and reactivity: Distinct similarities to whole proteins. J. Inorg. Biochem. 2016, 159, 29– 36,  DOI: 10.1016/j.jinorgbio.2016.02.016
      32
      Unbound position II in MXCXXC metallochaperone model peptides impacts metal binding mode and reactivity: Distinct similarities to whole proteins
      Shoshan, Michal S.; Dekel, Noa; Goch, Wojciech; Shalev, Deborah E.; Danieli, Tsafi; Lebendiker, Mario; Bal, Wojciech; Tshuva, Edit Y.
      Journal of Inorganic Biochemistry (2016), 159 (), 29-36CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)
      The effect of position II in the binding sequence of copper metallochaperones, which varies between Thr and His, was investigated through structural anal. and affinity and oxidn. kinetic studies of model peptides. A first Cys-Cu(I)-Cys model obtained for the His peptide at acidic and neutral pH, correlated with higher affinity and more rapid oxidn. of its complex; in contrast, the Thr peptide with the Cys-Cu(I)-Met coordination under neutral conditions demonstrated weaker and pH dependent binding. Studies with human antioxidant protein 1 (Atox1) and three of its mutants where S residues were replaced with Ala suggested that (a) the binding affinity is influenced more by the binding sequence than by the protein fold (b) pH may play a role in binding reactivity, and (c) mutating the Met impacted the affinity and oxidn. rate more drastically than did mutating one of the Cys, supporting its important role in protein function. Position II thus plays a dominant role in metal binding and transport.
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    33. 33
      Piątek, K.; Schwerdtle, T.; Hartwig, A.; Bal, W. Monomethylarsonous Acid Destroys a Tetrathiolate Zinc Finger Much More Efficiently than Inorganic Arsenite: Mechanistic Considerations and Consequences for DNA Repair Inhibition. Chem. Res. Toxicol. 2008, 21, 600– 606,  DOI: 10.1021/tx7003135
      33
      Monomethylarsonous Acid Destroys a Tetrathiolate Zinc Finger Much More Efficiently than Inorganic Arsenite: Mechanistic Considerations and Consequences for DNA Repair Inhibition
      Piatek, Katarzyna; Schwerdtle, Tanja; Hartwig, Andrea; Bal, Wojciech
      Chemical Research in Toxicology (2008), 21 (3), 600-606CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
      Arsenic compds. are human carcinogens. The ingested inorg. arsenic is metabolized to methylated derivs., which are considered to be more toxic than the inorg. species. Interactions of trivalent arsenicals with thiol groups of proteins are believed to be important for arsenic carcinogenesis, but inorg. arsenite appears to bind to thiol groups more strongly than the methylated AsIII species. Inhibition of the nucleotide excision repair pathway of DNA repair (NER) is likely to be of primary importance in arsenic carcinogenesis. Previously, we demonstrated that methylated AsIII compds. are more efficient than arsenite in releasing zinc from ZnXPAzf, the zinc finger of XPA, a crucial member of the NER complex. In this work, we used ESI-MS to compare aerobic reactivities of arsenite and monomethylarsonous acid (MMAIII) toward ZnXPAzf on the mol. level. We demonstrated that equimolar MMAIII released ZnII from ZnXPAzf easily, forming mono- and diarsenical derivs. of XPAzf. This reaction was accompanied by oxidn. of unprotected thiol groups of the monomethylarsinated peptide to intramol. disulfides. The estd. affinity of MMAIII to XPAzf is 30-fold higher than that established previously for arsenite binding to the thiol groups. No binding of arsenite to the thiol groups of XPAzf was obsd. under our exptl. conditions, and a 10-fold excess of arsenite was required to partially oxidize ZnXPAzf. These results indicate a particular susceptibility of tetrathiolate zinc fingers to MMAIII, thereby providing a novel mol. pathway in arsenic carcinogenesis.
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    34. 34
      Whittal, R. M.; Ball, H. L.; Cohen, F. E.; Burlingame, A. L.; Prusiner, S. B.; Baldwin, M. A. Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry. Protein Sci. 2000, 9, 332– 343,  DOI: 10.1110/ps.9.2.332
      34
      Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry
      Whittal, Randy M.; Ball, Haydn L.; Cohen, Fred E.; Burlingame, Alma L.; Prusiner, Stanley B.; Baldwin, Michael A.
      Protein Science (2000), 9 (2), 332-343CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)
      Electrospray ionization mass spectrometry (ESI-MS) was used to measure the binding of Cu2+ ions to synthetic peptides corresponding to sections of the sequence of the mature prion protein (PrP). ESI-MS demonstrates that Cu2- is unique among divalent metal ions in binding to PrP and defines the location of the major Cu2+ binding site as the octarepeat region in the N-terminal domain, contg. multiple copies of the repeat ProHisGlyGlyGlyTrpGlyGln. The stoichiometries of the complexes measured directly by ESI-MS are pH dependent: a peptide contg. four octarepeats chelates two Cu2+ ions at pH 6 but four at pH 7.4. At the higher pH, the binding of multiple Cu2+ ions occurs with a high degree of cooperativity for peptides C-terminally extended to incorporate a fifth histidine. Dissocn. consts. for each Cu2+ ion binding to the octarepeat peptides, reported here for the first time, are mostly in the low micromolar range; for the addn. of the third and fourth Cu2+ ions to the extended peptides at pH 7.4, KDs are <100 nM. N-terminal acetylation of the peptides caused some redn. in the stoichiometry of binding at both pHs. Cu2+ also binds to a peptide corresponding to the extreme N-terminus of PrP that precedes the octarepeats, arguing that this region of the sequence may also make a contribution to the Cu2+ complexation. Although the structure of the four-octarepeat peptide is not affected by pH changes in the absence of Cu2+, as judged by CD, Cu2+ binding induces a modest change at pH 6 and a major structural perturbation at pH 7.4. It is possible that PrP functions as a Cu2+ transporter by binding Cu2+ ions from the extracellular medium under physiol. conditions and then releasing some or all of this metal upon exposure to acidic pH in endosomes or secondary lysosomes.
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    35. 35
      Di Marco, V. B.; Bombi, G. G. Electrospray mass spectrometry (ESI-MS) in the study of metal-ligand solution equilibria. Mass Spectrom. Rev. 2006, 25, 347– 379,  DOI: 10.1002/mas.20070
      35
      Electrospray mass spectrometry (ESI-MS) in the study of metal-ligand solution equilibria
      Di Marco, Valerio B.; Bombi, G. Giorgio
      Mass Spectrometry Reviews (2006), 25 (3), 347-379CODEN: MSRVD3; ISSN:0277-7037. (John Wiley & Sons, Inc.)
      A review. In the 20 years, since the introduction of electrospray mass spectrometry (ESI-MS), the use of this technique in various fields of inorg., organometallic, and anal. chem. was steadily increasing. The application of ESI-MS to the study of metal-ligand soln. equil. is reviewed (till 2004 included). In a 1st section, advantages and drawbacks of ESI-MS in this type of application are described. Subsequently, a list of ∼300 studies is reported, in which ESI-MS was used to give no. and stoichiometry of the species at equil., or also to est. their stability consts. All studies are classified according to the metal ions under examn. Other related applications, such as host-guest interactions and metal ion-protein binding studies, are briefly reviewed as well.
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    36. 36
      Wyttenbach, T.; Liu, D.; Bowers, M. T. Interactions of the hormone oxytocin with divalent metal ions. J. Am. Chem. Soc. 2008, 130, 5993– 6000,  DOI: 10.1021/ja8002342
      36
      Interactions of the Hormone Oxytocin with Divalent Metal Ions
      Wyttenbach, Thomas; Liu, Dengfeng; Bowers, Michael T.
      Journal of the American Chemical Society (2008), 130 (18), 5993-6000CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The interaction of the cyclic nonapeptide oxytocin (OT) with a no. of alk. earth and divalent transition metal ions (X2+) was examd. employing mass spectrometry (MS) and ion mobility spectrometry (IMS) techniques in combination with mol. dynamics (MD) and d. functional theory (DFT) calcns. Under acidic conditions OT exhibits an exceptionally strong affinity for all divalent metal ions resulting in strong [OT + X]2+ peaks in the mass spectrum. Under basic conditions only Cu2+ and Ni2+-OT complexes were detected and these were singly, doubly, triply, or quadruply deprotonated. Collision-induced dissocn. of the [OT - 3H + Cu]- complex yielded exclusively C-terminal Cu2+-contg. fragments (Cu2+fragment3-), suggesting that the Cu2+ ligation site includes deprotonated C-terminal backbone amide nitrogen atoms and the N-terminal amino nitrogen atom in [OT - 3H + Cu]-. MD and DFT calcns. indicate a square-planar complex is consistent with these observations and with exptl. collision cross sections. MD and DFT calcns. also indicate either an octahedral or trigonal-bipyramidal complex between Zn2+ and OT is lowest in energy with carbonyl oxygens being the primary ligation sites. Both complexes yield cross sections in agreement with expt. The biol. impact of the structural changes induced in OT by divalent metal ion coordination is discussed.
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    37. 37
      Ikonomou, M. G.; Blades, A. T.; Kebarle, P. Investigations of the Electrospray Interface for Liquid Chromatography/Mass Spectrometry. Anal. Chem. 1990, 62, 957– 967,  DOI: 10.1021/ac00208a012
      37
      Investigations of the electrospray interface for liquid chromatography/mass spectrometry
      Ikonomou, Michael G.; Blades, Arthur T.; Kebarle, Paul
      Analytical Chemistry (1990), 62 (9), 957-67CODEN: ANCHAM; ISSN:0003-2700.
      The pos. ions, produced by electrospray from solns. of various analytes in methanol, were detected with an atm. pressure triple quadrupole mass spectrometer. Analytes studied included NH4+, Na+, K+, Cs+, Ca2+ and the onium ions BH+ of some 30 org. nitrogen bases B. The analyte ion sensitivities decrease with analyte ion concn. and presence of foreign electrolyte in the soln., at concn. above 10-5 mol/L. At low concns., sensitivities are very high such that ions at 10-8 mol/L concn. can be easily detected. Detection limits in the subfemtomol to attomol range have been achieved. The sensitivity of the org. bases B is pH dependent and increases as the [BH+] in soln. increases. However, the effect is obscured by the depression of the ion signal caused by foreign electrolyte. It is also shown that above 10-5 mol/L B in soln., gaseous B at sufficient pressure can be generated and gas-phase proton transfer to higher gas phase coanalytes can occur. Dimers, B2H+, may also be formed in the gas phase. The gas-phase ion reaction time is estd. as ∼2 ms.
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    38. 38
      Wortmann, A.; Kistler-Momotova, A.; Zenobi, R.; Heine, M. C.; Wilhelm, O.; Pratsinis, S. E. Shrinking droplets in electrospray ionization and their influence on chemical equilibria. J. Am. Soc. Mass Spectrom. 2007, 18, 385– 393,  DOI: 10.1016/j.jasms.2006.10.010
      38
      Shrinking Droplets in Electrospray Ionization and Their Influence on Chemical Equilibria
      Wortmann, Arno; Kistler-Momotova, Anna; Zenobi, Renato; Heine, Martin C.; Wilhelm, Oliver; Pratsinis, Sotiris E.
      Journal of the American Society for Mass Spectrometry (2007), 18 (3), 385-393CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)
      The authors investigated how chem. equil. are affected by the electrospray process, using simultaneous in situ measurements by laser-induced fluorescence (LIF) and phase Doppler anemometry (PDA). The motivation for this study was the increasing no. of publications in which electrospray ionization mass spectrometry is used for binding const. detn. The PDA was used to monitor droplet size and velocity, whereas LIF was used to monitor fluorescent analytes within the electrospray droplets. Using acetonitrile as solvent, the authors found an av. initial droplet diam. of 10 μm in the electrospray. The PDA allowed the authors to follow the evolution of these droplets down to a size of 1 μm. Rhodamine B-sulfonylchloride was used as a fluorescent analyte within the electrospray. By spatially resolved LIF it was possible to probe the dimerization equil. of this dye. Measurements at different spray positions showed no influence of the decreasing droplet size on the monomer-dimer equil. However, with the fluorescent dye pair DCM and oxazine 1 it was shown that a concn. increase does occur within electrosprayed droplets, using fluorescence resonance energy transfer as a probe for the av. pair distance.
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    39. 39
      Kebarle, P.; Tang, L. From ions in solution to ions in the gas phase-the mechanism of electrospray mass spectrometry. Anal. Chem. 1993, 65, 972A– 986A,  DOI: 10.1021/ac00070a715
      39
      From ions in solution to ions in the gas phase - the mechanism of electrospray mass spectrometry
      Kebarle, Paul; Tang, Liang
      Analytical Chemistry (1993), 65 (22), 972A-986ACODEN: ANCHAM; ISSN:0003-2700.
      The title topic is reviewed with 44 refs. The subjects include: the electrospray (ES) mechanism, prodn. of charged droplets at the ES capillary tip, shrinkage of charged ES droplets, nature of processes leading to formation of gas-phase ions, details of the Iribarne ion evapn. theory, dependence of ion intensities on concn., effects due to the addn. of 2 electrolytes to the solvent, comparison of coeffs. with Iribarne theory and SIDT (single ion in droplet theory), emission of gas-phase ions from the Taylor tip of the ES capillary, and formation mechanisms of multiply-charged macroions.
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    40. 40
      Wilkins, R. G. Kinetics and mechanism of reactions of transition metal complexes, 2nd ed.; VCH: Weinheim, online resource, 2003.
      There is no corresponding record for this reference.
    41. 41
      Teng, X.; Stefaniak, E.; Girvan, P.; Kotuniak, R.; Płonka, D.; Bal, W.; Ying, L. Hierarchical binding of copperII to N-truncated Aβ4–16 peptide. Metallomics 2020, 12, 470– 473,  DOI: 10.1039/C9MT00299E
      41
      Hierarchical binding of copperII to N-truncated Aβ4-16 peptide
      Teng, Xiangyu; Stefaniak, Ewelina; Girvan, Paul; Kotuniak, Radoslaw; Plonka, Dawid; Bal, Wojciech; Ying, Liming
      Metallomics (2020), 12 (4), 470-473CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)
      N-Truncated Aβ4-42 displays a high binding affinity with CuII. A mechanistic scheme of the interactions between Aβ4-42 and CuII has been proposed using a fluorescence approach. The timescales of different conversion steps were detd. This kinetic mechanism indicates the potential synaptic functions of Aβ4-42 during neurotransmission.
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    42. 42
      Matsumoto, A.; Fukumoto, T.; Adachi, H.; Watarai, H. Electrospray ionization mass spectrometry of metal complexes. Gas phase formation of a binuclear copper(II)-5-Br-PADAP complex. Anal. Chim. Acta 1999, 390, 193– 199,  DOI: 10.1016/S0003-2670(99)00222-6
      42
      Electrospray ionization mass spectrometry of metal complexes. Gas phase formation of a binuclear copper(II)-5-Br-PADAP complex
      Matsumoto, Atsushi; Fukumoto, Takao; Adachi, Hiroshi; Watarai, Hitoshi
      Analytica Chimica Acta (1999), 390 (1-3), 193-200CODEN: ACACAM; ISSN:0003-2670. (Elsevier Science B.V.)
      The complexes formed from Cu(II) and 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (5-Br-PADAP or HL) in aq. MeOH soln. was studied by electrospray ionization mass spectrometry. The soln. of a 1:1 complex of Cu(II) with 5-Br-PADAP showed five peaks assignable to binuclear [Cu2L2(AcO)]+ and mononuclear [CuL]+, [CuL(H2O)]+, [CuL(AcOH)]+ and [CuL(HL)]+ (AcO = acetate). Collision activated dissocn. revealed the relative order of bonding strengths; Cu-L > Cu-HL > CuL-AcOH > CuL-H2O. The peak intensities of the binuclear complex showed 2nd-order dependency on those of the mono complex. As for the soln. of Ni(II)-5-Br-PADAP, no binuclear complex was obsd. in the mass spectra. Thus, [Cu2L2(AcO)]+ was probably formed by the fast gas phase reaction: 2[CuL]++AcO-↹[Cu2L2(AcO)]+.
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    43. 43
      Dill, K. A. Dominant forces in protein folding. Biochemistry 1990, 29, 7133– 7155,  DOI: 10.1021/bi00483a001
      43
      Dominant forces in protein folding
      Dill, Ken A.
      Biochemistry (1990), 29 (31), 7133-55CODEN: BICHAW; ISSN:0006-2960.
      A review, with 328 refs., on the nature and magnitudes of dominant forces in protein folding. Contributions to free energy of folding arising from electrostatics, H bonding and van der Waals interactions, intrinsic propensities, and hydrophobic interactions are explored. Also considered are the opposing forces, conformational entropy and electrostatics.
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    44. 44
      Doonan, S. Peptides and proteins; Royal Society of Chemistry: Cambridge, 2002.
      There is no corresponding record for this reference.
    45. 45
      Zhou, S.; Cook, K. D. Protonation in electrospray mass spectrometry: Wrong-way-round or right-way-round?. J. Am. Soc. Mass Spectrom. 2000, 11, 961– 966,  DOI: 10.1016/S1044-0305(00)00174-4
      45
      Protonation in electrospray mass spectrometry: wrong-way-round or right-way-round?
      Zhou, Shaolian; Cook, Kelsey D.
      Journal of the American Society for Mass Spectrometry (2000), 11 (11), 961-966CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Science Inc.)
      The term "wrong-way-round ionization" has been used in studies of electrospray ionization to describe the observation of protonated or deprotonated ions when sampling strongly basic or acidic solns. (resp.) where such ions are not expected to exist in appreciable concns. in soln. Study of the dependence of ionization of the weak base caffeine on the electrospray capillary potential reveals three distinct contributors to wrong-way-round ionization. At near-neutral pH in solns. of low ionic strength, protonation of caffeine results from the surface enrichment of electrolytically produced protons in the surface layer of the droplets from which ions are desorbed. For solns. made strongly basic with ammonia, gas-phase proton transfer from ammonium ions can create protonated caffeine. These two mechanisms have been discussed previously elsewhere. For solns. of high ionic strength at neutral or high pH, the data suggest that discharge-induced ionization is responsible for the prodn. of protonated caffeine. This mechanism probably accounts for some of the wrong-way-round ionization reported elsewhere.
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    46. 46
      Wu, Q.; Gao, J.; Joseph-McCarthy, D.; Sigal, G. B.; Bruce, J. E.; Whitesides, G. M.; Smith, R. D. Carbonic Anhydrase-Inhibitor Binding: From Solution to the Gas Phase. J. Am. Chem. Soc. 1997, 119, 1157– 1158,  DOI: 10.1021/ja9630250
      46
      Carbonic Anhydrase-Inhibitor Binding: From Solution to the Gas Phase
      Wu, Qinyuan; Gao, Jinming; Joseph-McCarthy, Diane; Sigal, George B.; Bruce, James E.; Whitesides, George M.; Smith, Richard D.
      Journal of the American Chemical Society (1997), 119 (5), 1157-1158CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The binding of para-substituted benzenesulfonamide inhibitors to bovine carbonic anhydrase II (BCAII, EC 4.2.1.1) in the gas phase and in soln. has been studied by electrospray ionization-mass spectrometry and fluorescence spectroscopy, resp. The off-rates of BCAII-inhibitor complexes in soln. are primarily detd. by the hydrophobic interactions between the inhibitor and the enzyme, while their corresponding gas phase stabilities are governed by the polar surface interactions. The results provide insights into the factors governing gas phase stability of the charged complexes, and show that relative stabilities in soln. and the gas phase are substantially different.
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    47. 47
      Wales, T. E.; Engen, J. R. Partial unfolding of diverse SH3 domains on a wide timescale. J. Mol. Biol. 2006, 357, 1592– 1604,  DOI: 10.1016/j.jmb.2006.01.075
      47
      Partial unfolding of diverse SH3 domains on a wide timescale
      Wales, Thomas E.; Engen, John R.
      Journal of Molecular Biology (2006), 357 (5), 1592-1604CODEN: JMOBAK; ISSN:0022-2836. (Elsevier B.V.)
      SH3 domains are small, modular domains that are found in many proteins, esp. signal transduction proteins such as tyrosine kinases. While much is known about the sequences and tertiary structures of SH3 domains, far less is known about their soln. dynamics. A slow, partial unfolding event that occurs under physiol. conditions was previously identified in the Hck SH3 domain using H-exchange (HX) mass spectrometry (MS). To det. if this unfolding was unique to Hck SH3, HX MS was used to analyze 11 other SH3 domains: 7 SH3 domains from Src-family kinases and 5 SH3 domains from various proteins. A wide variety of unfolding rates were found, with unfolding half-lives ranging from 1 s to 1 h. The Lyn and α-spectrin SH3 domains exhibited slow, partial unfolding in β-strands D and E and part of the RT-loop. Hck SH3 also underwent partial unfolding in the same region, implying that a unique feature in this area of the domains is responsible for the partial unfolding. Partial unfolding was, however, not a function of sequence conservation. Although the Fyn and Yes SH3 domains are very similar to Hck SH3 in sequence, they exhibited no evidence of partial unfolding. Overall, the results suggest that while the tertiary structure of SH3 domains is highly conserved, the dynamics of SH3 domains are variable.
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    48. 48
      Orte, A.; Craggs, T. D.; White, S. S.; Jackson, S. E.; Klenerman, D. Evidence of an intermediate and parallel pathways in protein unfolding from single-molecule fluorescence. J. Am. Chem. Soc. 2008, 130, 7898– 7907,  DOI: 10.1021/ja709973m
      48
      Evidence of an Intermediate and Parallel Pathways in Protein Unfolding from Single-Molecule Fluorescence
      Orte, Angel; Craggs, Timothy D.; White, Samuel S.; Jackson, Sophie E.; Klenerman, David
      Journal of the American Chemical Society (2008), 130 (25), 7898-7907CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Detg. how proteins fold into their native structures is a subject of great importance, since ultimately it will allow protein structure and function to be predicted from primary sequence data. In addn., there is now a clear link between protein unfolding and misfolding events and many disease states. However, since proteins fold over rugged, multidimensional energy landscapes, this is a challenging exptl. and theor. problem. Single-mol. fluorescence methods developed over the past decade have the potential to follow the unfolding/folding of individual mols. Mapping out the landscape without ensemble averaging will enable the identification of intermediate states which may not be significantly populated, in addn. to the presence of multiple pathways. To date, there have been only a limited no. of single-mol. folding/unfolding studies under nonequil. conditions and no intermediates have been obsd. Here, for the first time, we present a single-mol. study of the unfolding of a large autofluorescent protein, Citrine, a variant of green fluorescent protein. Single-mol. fluorescence techniques are used to directly detect an intermediate on the unfolding/folding pathway and the existence of parallel unfolding pathways. This work, and the novel methods used, shows that single-mol. fluorescence can now provide new, hitherto exptl. inaccessible, insights into the folding/unfolding of proteins.
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    49. 49
      Kubelka, J.; Eaton, W. A.; Hofrichter, J. Experimental Tests of Villin Subdomain Folding Simulations. J. Mol. Biol. 2003, 329, 625– 630,  DOI: 10.1016/S0022-2836(03)00519-9
      49
      Experimental Tests of Villin Subdomain Folding Simulations
      Kubelka, Jan; Eaton, William A.; Hofrichter, James
      Journal of Molecular Biology (2003), 329 (4), 625-630CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Science Ltd.)
      We have used laser temp.-jump to investigate the kinetics and mechanism of folding the 35 residue subdomain of the villin headpiece. The relaxation kinetics are biphasic with a sub-microsecond phase corresponding to a helix-coil transition and a slower microsecond phase corresponding to overall unfolding/refolding. At 300 K, the folding time is 4.3(±0.6) μs, making it the fastest folding, naturally occurring protein, with a rate close to the theor. speed limit. This time is in remarkable agreement with the prediction of 5 (+11,-3) μs by Zagrovic et al. from atomistic mol. dynamics simulations using an implicit solvent model. We test their prediction that replacement of the C-terminal phenylalanine residue with alanine will increase the folding rate by removing a transient non-native interaction. We find that the alanine substitution has no effect on the folding rate or on the equil. const. Implications of this result for the validity of the simulated folding mechanism are discussed.
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    50. 50
      Trapaidze, A.; Hureau, C.; Bal, W.; Winterhalter, M.; Faller, P. Thermodynamic study of Cu2+ binding to the DAHK and GHK peptides by isothermal titration calorimetry (ITC) with the weaker competitor glycine. JBIC, J. Biol. Inorg. Chem. 2012, 17, 37– 47,  DOI: 10.1007/s00775-011-0824-5
      50
      Thermodynamic study of Cu2+ binding to the DAHK and GHK peptides by isothermal titration calorimetry (ITC) with the weaker competitor glycine
      Trapaidze, Ana; Hureau, Christelle; Bal, Wojciech; Winterhalter, Mathias; Faller, Peter
      JBIC, Journal of Biological Inorganic Chemistry (2012), 17 (1), 37-47CODEN: JJBCFA; ISSN:0949-8257. (Springer)
      The peptides Asp-Ala-His-Lys (DAHK) and Gly-His-Lys (GHK) are naturally occurring Cu(II)-chelating motifs in human serum and cerebrospinal fluid. Here, the sensitive thermodn. technique isothermal titrn. calorimetry was used to study the energetics of Cu(II) binding to DAHK and GHK peptides in the presence of the weaker ligand glycine as a competitor. DAHK and GHK bind Cu(II) predominantly in a 1:1 stoichiometry with conditional dissocn. consts. [i.e., at pH 7.4, in the absence of the competing chelators glycine and 2-(4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid buffer] of 2.6 ± 0.4 × 10-14 M and 7.0 ± 1.0 × 10-14 M, resp. Furthermore, the apparent ΔH values were measured and the no. of protons released upon Cu(II) binding was detd. by performing expts. in different buffers. This allowed us to det. the conditional ΔG, ΔH, and ΔS, i.e., cor. for the contributions of the weaker ligand glycine and the buffer at pH 7.4. We found that the entropic and enthalpic contributions to the Cu(II) binding to GHK and DAHK are distinct, with a enthalpic contribution for GHK. The thermodn. parameters obtained correspond well to those in the literature obtained by other techniques, suggesting that the use of the weaker ligand glycine as a competitor in isothermal titrn. calorimetry provides accurate data for Cu(II) binding to high-affinity peptides, which cannot be accurately detd. without the use of a competitor ligand.
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    51. 51
      North, M. L.; Wilcox, D. E. Shift from Entropic Cu2+ Binding to Enthalpic Cu+ Binding Determines the Reduction Thermodynamics of Blue Copper Proteins. J. Am. Chem. Soc. 2019, 141, 14329– 14339,  DOI: 10.1021/jacs.9b06836
      51
      Shift from Entropic Cu2+ Binding to Enthalpic Cu+ Binding Determines the Reduction Thermodynamics of Blue Copper Proteins
      North, Molly L.; Wilcox, Dean E.
      Journal of the American Chemical Society (2019), 141 (36), 14329-14339CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The enthalpic and entropic components of Cu2+ and Cu+ binding to the blue copper protein azurin have been quantified with isothermal titrn. calorimetry (ITC) measurements and anal., providing the first such exptl. values for Cu+ binding to a protein. The high affinity of azurin for Cu2+ is entirely due to a very favorable binding entropy, while its even higher affinity for Cu+ is due to a favorable binding enthalpy and entropy. The binding thermodn. provide insight into bond enthalpies at the blue copper site and entropic contributions from desolvation and proton displacement. These values were used in thermodn. cycles to det. the enthalpic and entropic contributions to the free energy of redn. and thus the redn. potential. The redn. thermodn. obtained with this method are in good agreement with previous results from temp.-dependent electrochem. measurements. The calorimetry method, however, provides new insight into contributions from the initial (oxidized) and final (reduced) states of the redn. Since ITC measurements quantify the protons that are displaced upon metal binding, the proton transfer that is coupled with electron transfer is also detd. with this method. Preliminary results for Cu2+ and Cu+ binding to the Phe114Pro variant of azurin demonstrate the insight about protein tuning of the redn. potential that is provided by the binding thermodn. of each metal oxidn. state.
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    52. 52
      Konermann, L. Addressing a Common Misconception: Ammonium Acetate as Neutral pH “Buffer” for Native Electrospray Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2017, 28, 1827– 1835,  DOI: 10.1007/s13361-017-1739-3
      52
      Addressing a Common Misconception: Ammonium Acetate as Neutral pH "Buffer" for Native Electrospray Mass Spectrometry
      Konermann, Lars
      Journal of the American Society for Mass Spectrometry (2017), 28 (9), 1827-1835CODEN: JAMSEF; ISSN:1044-0305. (Springer)
      A review and discussion. Native ESI-MS involves the transfer of intact proteins and biomol. complexes from soln. into the gas phase. One potential pitfall is the occurrence of pH-induced changes that can affect the analyte while it is still surrounded by solvent. Most native ESI-MS studies employ neutral aq. ammonium acetate solns. It is a widely perpetuated misconception that ammonium acetate buffers the analyte soln. at neutral pH. By definition, a buffer consists of a weak acid and its conjugate weak base. The buffering range covers the weak acid pKa ± 1 pH unit. NH4+ and CH3-COO- are not a conjugate acid/base pair, which means that they do not constitute a buffer at pH 7. Dissoln. of ammonium acetate salt in water results in pH 7, but this pH is highly labile. Ammonium acetate does provide buffering around pH 4.75 (the pKa of acetic acid) and around pH 9.25 (the pKa of ammonium). This implies that neutral ammonium acetate solns. electrosprayed in pos. ion mode will likely undergo acidification down to pH 4.75 ± 1 in the ESI plume. Ammonium acetate nonetheless remains a useful additive for native ESI-MS. It is a volatile electrolyte that can mimic the solvation properties experienced by proteins under physiol. conditions. Also, a drop from pH 7 to around pH 4.75 is less dramatic than the acidification that would take place in pure water. It is hoped that the habit of referring to pH 7 solns. as ammonium acetate "buffer" will disappear from the literature. Ammonium acetate "soln." should be used instead.
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    53. 53
      Raamat, E.; Kaupmees, K.; Ovsjannikov, G.; Trummal, A.; Kütt, A.; Saame, J.; Koppel, I.; Kaljurand, I.; Lipping, L.; Rodima, T.; Pihl, V.; Koppel, I. A.; Leito, I. Acidities of strong neutral Brønsted acids in different media. J. Phys. Org. Chem. 2013, 26, 162– 170,  DOI: 10.1002/poc.2946
      53
      Acidities of strong neutral Bronsted acids in different media
      Raamat, Elin; Kaupmees, Karl; Ovsjannikov, Gea; Trummal, Aleksander; Kuett, Agnes; Saame, Jaan; Koppel, Ivar; Kaljurand, Ivari; Lipping, Lauri; Rodima, Toomas; Pihl, Viljar; Koppel, Ilmar A.; Leito, Ivo
      Journal of Physical Organic Chemistry (2013), 26 (2), 162-170CODEN: JPOCEE; ISSN:0894-3230. (John Wiley & Sons Ltd.)
      Acidities of different families of acids are examd. in media of different phys. and chem. nature: water, acetonitrile (AN), 1,2-dichloroethane (DCE) and the gas phase, with special emphasis on strong acids. Included are OH (carboxylic acids, alcs., and phenols), NH (sulfonamides, imides), and CH (phenylmalononitriles, etc.) acids as well as HCl, HBr, and HI. Dependence of the acidity trends on moving from water to the gas phase on the nature of the acidity center, and the mol. structure are analyzed. The acidity orders are different in water, AN, DCE, and the gas phase. In some cases the differences are dramatic. AN and DCE display similar acidity order in the set of the studied acids. The decisive factor for the behavior of the acids when transferring between different media is the extent of charge delocalization in the anion and the recently introduced weighted av. pos. sigma parameter in spite of its simplicity enables interpretation of the trends in the majority of cases. Copyright © 2012 John Wiley and Sons, Ltd.
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    54. 54
      Wang, H.; Agnes, G. R. Kinetically labile equilibrium shifts induced by the electrospray process. Anal. Chem. 1999, 71, 4166– 4172,  DOI: 10.1021/ac981375u
      54
      Kinetically Labile Equilibrium Shifts Induced by the Electrospray Process
      Wang, Hongjun; Agnes, George R.
      Analytical Chemistry (1999), 71 (19), 4166-4172CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      The complexation reactions between the alk. earth metal ions and EDTA were studied by electrospray mass spectrometry to measure the change in concn. of the metal ion-EDTA complex (MY2-) in the gas phase relative to the soln.-phase equil. concn. This work focused on the soln. pH range from 4 to 7 where there exists free metal ions in soln. at equil. The equil. shift, measured through quantitation of the increased abundance of the MY2- species in the gas phase, was largest for barium and smallest for magnesium. The cause of the net equil. shift of the MY2- species is the combined effect of an electrolytic increase in pH within the capillary plus an addnl. shift within the evapg. droplets. In a thin diffusion-limited layer created by the products of electrolysis mixing with the bulk soln. at the ES capillary tip, the labile species reequilibrate at a new, higher pH. In the evapg. droplets, the formation of new labile species due to increased solute concns. is kinetically controlled because the ion residence time in the droplet prior to desorption is only ∼5 μs. These results are briefly discussed with respect to the potential for utilizing electrospray mass spectrometry for kinetically labile equil. studies.
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    55. 55
      McDonald, L. W.; Campbell, J. A.; Clark, S. B. Failure of ESI spectra to represent metal-complex solution composition: A study of lanthanide-carboxylate complexes. Anal. Chem. 2014, 86, 1023– 1029,  DOI: 10.1021/ac401751r
      55
      Failure of ESI Spectra to Represent Metal-Complex Solution Composition: A Study of Lanthanide-Carboxylate Complexes
      McDonald, Luther W.; Campbell, James A.; Clark, Sue B.
      Analytical Chemistry (Washington, DC, United States) (2014), 86 (2), 1023-1029CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Electrospray ionization-mass spectrometry (ESI-MS) shows great promise as a rapid method to identify metal-ligand complexes in soln. However, its application for quant. detn. of the distribution of species present in complicated equil. is still in its infancy, and a direct correlation between ions obsd. in the gas phase and species expected in soln. must be made with caution. The present work focuses on a seemingly simple system; the complexation of lanthanide cations with the acetate ligand. Using a high resoln. quadrupole time-of-flight mass spectrometer, ions created by electrospray of solns. contg. trivalent Nd and acetate were identified. The gas phase distribution of species was compared to the soln. phase speciation predicted using thermodn. complexation consts. Apparent gas phase speciation diagrams were constructed as a function of soln. conditions and fragmentation potential. Despite the expected variability of metal-ligand complexes as soln. conditions change, the obsd. gas phase speciation was independent of the metal to ligand ratio but dependent on the operating conditions of the ESI-MS.
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    56. 56
      Zhang, M.; Gumerov, D. R.; Kaltashov, I. A.; Mason, A. B. Indirect detection of protein-metal binding: Interaction of serum transferrin with In3+ and Bi3+. J. Am. Soc. Mass Spectrom. 2004, 15, 1658– 1664,  DOI: 10.1016/j.jasms.2004.08.009
      56
      Indirect detection of protein-Metal binding: Interaction of serum transferrin with In3+ and Bi3+
      Zhang, Mingxuan; Gumerov, Dmitry R.; Kaltashov, Igor A.; Mason, Anne B.
      Journal of the American Society for Mass Spectrometry (2004), 15 (11), 1658-1664CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)
      Transferrins comprise a class of monomeric glycoproteins found in all vertebrates, whose function is iron sequestration and transport. In addn. to iron, serum transferrin also binds a variety of other metals and is believed to provide a route for the in vivo delivery of such metals to cells. In the present study, ESI MS is used to investigate interactions between human serum transferrin and two nonferrous metals, indium (a commonly used imaging agent) and bismuth (a component of many antiulcer drugs). While the UV-Vis absorption spectroscopy measurements clearly indicate that both metals bind strongly to transferrin in soln., the metal-protein complex can be detected by ESI MS only for indium, but not for bismuth. Despite the apparently low stability of the transferrin-bismuth complex in the gas phase, presence of such complex in soln. can be established by ESI MS indirectly. This is done by monitoring the evolution of charge state distributions of transferrin ions upon acid-induced protein unfolding in the presence and in the absence of the metal in soln. The anomalous instability of the transferrin-bismuth complex in the gas phase is rationalized in terms of conformational differences between this form of transferrin and the holo-forms of this protein produced by binding of metals with smaller ionic radii (e.g., Fe3+ and In3+). The large size of Bi3+ ion is likely to prevent formation of a closed conformation (canonical structure of the holo-protein), resulting in a non-native metal coordination. It is suggested that transferrin retains the open conformation (characteristic of the apo-form) upon binding Bi3+, with only two ligands in the metal coordination sphere provided by the protein itself. This suggestion is corroborated by the results of CD measurements in the near-UV range. Since the cellular consumption of metals in the transferrin cycle critically depends upon recognition of the holo-protein complex by the transferrin receptor, the noncanonical conformation of the transferrin-bismuth complex may explain very inefficient delivery of bismuth to cells even when a high dosage of bismuth-contg. drugs is administered for prolonged periods of time.
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    57. 57
      Gatlin, C. L.; Turecek, F.; Vaisar, T. Determination of soluble Cu (I) and Cu (II) species in jet fuel by electrospray ionization mass spectrometry. Anal. Chem. 1994, 66, 3950– 3958,  DOI: 10.1021/ac00094a016
      57
      Determination of Soluble Cu(I) and Cu(II) Species in Jet Fuel by Electrospray Ionization Mass Spectrometry
      Gatlin, Christine L.; Turecek, Frantisek; Vaisar, Tomas
      Analytical Chemistry (1994), 66 (22), 3950-8CODEN: ANCHAM; ISSN:0003-2700.
      Sol. Cu(I) and Cu(II) species in jet fuel (e.g., formed by exposure of jet fuels to Cu surfaces and extd. by metal deactivator additives) were analyzed either by electrospray-ionization mass spectrometry combined with online liq. chromatog. on poly(vinyl alc.) sorbent or by off-line solvent extn. Detection limits in the 30-150 ppb range were achieved for Cu2+DMD (Cu2+ complex with N,N'-disalicylidene-1,2-propylenediamine), which was detected by mass spectrometry as its protonated ion. Low ppb detection limits were possible for the Na+ adduct. Collisional activation of Cu2+ carboxylate complexes in the gas phase induces intramol. electron transfer, resulting in redn. of the metal ion to Cu+ with concomitant ligand oxidn. and elimination.
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    58. 58
      Lavanant, H.; Hoppilliard, Y. Formation and fragmentation of α-amino acids complexed by Cu+. J. Mass Spectrom. 1997, 32, 1037– 1049,  DOI: 10.1002/(SICI)1096-9888(199711)32:10<1037::AID-JMS556>3.0.CO;2-L
      58
      Formation and fragmentation of α-amino acids complexed by Cu+
      Lavanant, Helene; Hoppilliard, Yannik
      Journal of Mass Spectrometry (1997), 32 (10), 1037-1049CODEN: JMSPFJ; ISSN:1076-5174. (Wiley)
      Gas phase complexes of α-amino acids with Cu+ have been engendered in a plasma desorption ion source by bombarding a mixt. of α-amino acids and cupric salts. The resulting adducts MCu+ and the fragments that include copper were studied using a time-of-flight analyzer. The main fragments result from a loss of 46 u, this fragmentation being very similar to the one obsd. for protonated mols. One noticeable exception, however, is arginine and lysine which give very little loss of 46 u as protonated mols. but do give a significant amt. of it when cationized by Cu+. The study of valine-d, has shown that the migration accompanying the fragmentations principally involves the hydrogens linked to heteroatoms. The deuteration of exchangeable hydrogens, done on several amino acids, confirmed these results. This fact, plus evidence of an interaction between Cu+ and the side chain of non-aliph. α-amino acids, has led to the suggestion of several mechanisms.
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    59. 59
      Lavanant, H.; Virelizier, H.; Hoppilliard, Y. Reduction of copper (II) complexes by electron capture in an electrospray ionization source. J. Am. Soc. Mass Spectrom. 1998, 9, 1217– 1221,  DOI: 10.1016/S1044-0305(98)00100-7
      59
      Reduction of copper(II) complexes by electron capture in an electrospray ionization source
      Lavanant, Helene; Virelizier, Henri; Hoppilliard, Yannik
      Journal of the American Society for Mass Spectrometry (1998), 9 (11), 1217-1221CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Science Inc.)
      The relative proportion of 1:1 Cu(I)- and Cu(II)-peptide complexes, PeptCu(I)+ and [Pept - H+Cu(II)]+, yielded by electrospray ionization of copper sulfate and peptide, H-Gly-His-Lys-OH, solns. in H2O/MeOH was examd. under different source conditions. Two factors leading to an increase in Cu(I):complex ratio were found. First, the increase of nozzle-skimmer voltages caused collision-induced dissocn. of Cu(II) complexes, and most probably, this favored ligand-to-metal electron transfers that resulted in the decoordination of oxydated ligands to form PeptCu+. Second, independent of these "inner sphere" processes that involve only electron exchange inside the coordination sphere around the metal cation, an increase in source voltages with a concomitant increase of current and, supposedly, electron counterflow between the counterelectrode and the capillary caused an increase in PeptCu+ relative proportion. The hypothesis that an "outer sphere" electron capture might happen in these conditions was verified by using discharge suppressing SF6 gas as nebulizing gas. The electroneg. gas reduced the current brought on by high voltages and inhibited the PeptCu+ increase phenomenon.
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    60. 60
      Tsybizova, A.; Roithová, J. Copper-catalyzed reactions: Research in the gas phase. Mass Spectrom. Rev. 2016, 35, 85– 110,  DOI: 10.1002/mas.21464
      60
      Copper-catalyzed reactions: Research in the gas phase
      Tsybizova, Alexandra; Roithova, Jana
      Mass Spectrometry Reviews (2016), 35 (1), 85-110CODEN: MSRVD3; ISSN:0277-7037. (John Wiley & Sons, Inc.)
      Electrospray ionization mass spectrometry (ESI-MS) is becoming an important tool for mechanistic studies in org. and organometallic chem. It allows investigation of reaction mixts. including monitoring of reactants, products, and intermediates, studying properties of the intermediates and their reactivity. Studying the reactive species in the gas phase can be advantageously combined with theor. calcns. This review is focused on ESI-MS studies of copper-catalyzed reactions. Possible effects of the electrospray process on the transfer of the copper complexes to the gas phase are discussed. The plethora of mass spectrometric approaches is demonstrated on copper mediated C-H activations, cross coupling reactions, rearrangements, organocuprate chem., and other examples.
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    61. 61
      Di Marco, V. B.; Bombi, G. G.; Zambon, S.; Traldi, P. Metal-ligand solution equilibria studied by electrospray ionization mass spectrometry: Effect of instrumental parameters. J. Mass Spectrom. 2009, 44, 120– 127,  DOI: 10.1002/jms.1481
      61
      Metal-ligand solution equilibria studied by electrospray ionization mass spectrometry: effect of instrumental parameters
      Di Marco, Valerio B.; Bombi, G. Giorgio; Zambon, Stefano; Traldi, Pietro
      Journal of Mass Spectrometry (2009), 44 (1), 120-127CODEN: JMSPFJ; ISSN:1076-5174. (John Wiley & Sons Ltd.)
      Electrospray ionization mass spectrometry (ESI-MS) is being increasingly employed in the study of metal-ligand equil. in aq. soln. In the present work, the ESI-MS spectral changes due to different settings of the following instrumental parameters are analyzed: the soln. flow rate (FS), the nebulizer gas flow rate (FG), the sprayer potential (E), and the temp. of the entrance capillary (T). Twenty-eight spectra were obtained for each of six samples contg. aluminum(III) and 2,3-dihydroxypyridine at various pH, in the absence or in the presence of a buffer and of sodium ions. Among the considered instrumental parameters, T produced the largest effects on the ionic intensities. FS and FG affected the ESI-MS spectra to a lower extent than T. In the investigated conditions E had the weakest effects on the spectra. The correlations obsd. between the ionic intensities and these instrumental parameters were interpreted considering the presence of three kinds of perturbations occurring in the ESI-MS ion source: formation of some dimers in the droplets, different transfer efficiencies from the droplets to the gas phase for different complexes (according to their surface activity), and subsequent partial thermal decompn. of the dimers and of one of the monomeric complexes in the gas phase. The results obtained show that the evaluation of the effects produced in the ESI-MS spectra by a change of instrumental parameters can allow to identify the perturbations occurring when metal-ligand solns. are studied by ESI-MS.
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    62. 62
      Kotuniak, R.; Frączyk, T.; Skrobecki, P.; Płonka, D.; Bal, W. Gly-His-Thr-Asp-Amide, an Insulin-Activating Peptide from the Human Pancreas Is a Strong Cu(II) but a Weak Zn(II) Chelator. Inorg. Chem. 2018, 57, 15507– 15516,  DOI: 10.1021/acs.inorgchem.8b02841
      62
      Gly-His-Thr-Asp-Amide, an Insulin-Activating Peptide from the Human Pancreas Is a Strong Cu(II) but a Weak Zn(II) Chelator
      Kotuniak, Radoslaw; Fraczyk, Tomasz; Skrobecki, Piotr; Plonka, Dawid; Bal, Wojciech
      Inorganic Chemistry (2018), 57 (24), 15507-15516CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
      The Cu(II) and Zn(II) binding abilities of Gly-His-Thr-Asp-amide (GHTD-am), a tetrapeptide co-released from the pancreas along with insulin, were studied using UV-vis and CD spectroscopies, potentiometry, and calorimetry. GHTD-am is a very strong Cu(II) chelator, forming a three-nitrogen complex with a conditional affinity const. CK at pH 7.4 of 4.5 × 1012 M-1. The fourth coordination site can be occupied by a solvent mol. or a ternary ligand, such as imidazole, with CK on the order of several hundred reciprocal molar. The Zn(II) binding ability of GHTD-am is relatively weak, with CK values at pH 7.4 of 3.0 × 104 and 2.0 × 103 M-1 for the first and second GHTD-am mol. coordinated, resp. These results are discussed in light of the modes of interactions of Zn(II) and Cu(II) ions with insulin. A direct effect of GHTD-am on the Zn(II) interactions with insulin is unlikely, but its Cu(II) complex may have a biol. relevance because of its high affinity and ability to form ternary complexes.
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    63. 63
      Płonka, D.; Bal, W. The N-terminus of hepcidin is a strong and potentially biologically relevant Cu(II) chelator. Inorg. Chim. Acta 2018, 472, 76– 81,  DOI: 10.1016/j.ica.2017.06.051
      63
      The N-terminus of hepcidin is a strong and potentially biologically relevant Cu(II) chelator
      Plonka, Dawid; Bal, Wojciech
      Inorganica Chimica Acta (2018), 472 (), 76-81CODEN: ICHAA3; ISSN:0020-1693. (Elsevier B.V.)
      Hepcidin is an iron regulatory hormone, involved also in immune response in vertebrates. It contains the N-terminal Asp-Thr-His site able to bind Cu(II) ions. A significant discrepancy exist in the literature regarding Cu(II) affinity of this site in hepcidin. Our study focused on the model DTHFPI-NH2 hexapeptide reflecting the N-terminal motif of mature hepcidin. Using potentiometry, UV-vis and CD spectroscopies we demonstrated that DTHFPI-NH2 is the strongest Cu(II) binding peptide discovered so far. A competition assay with human serum albumin (HSA) confirmed this high affinity and demonstrated that DTHFPI-NH2. withdraws all Cu(II) from HSA under equimolar concns. Based on these results we propose that hepcidin could exist as Cu(II) complex in blood, esp. under inflammatory conditions, when its serum concn. is elevated.
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    64. 64
      Bossak, K.; Drew, S. C.; Stefaniak, E.; Płonka, D.; Bonna, A.; Bal, W. The Cu(II) affinity of the N-terminus of human copper transporter CTR1: Comparison of human and mouse sequences. J. Inorg. Biochem. 2018, 182, 230– 237,  DOI: 10.1016/j.jinorgbio.2018.01.011
      64
      The Cu(II) affinity of the N-terminus of human copper transporter CTR1: Comparison of human and mouse sequences
      Bossak, Karolina; Drew, Simon C.; Stefaniak, Ewelina; Plonka, Dawid; Bonna, Arkadiusz; Bal, Wojciech
      Journal of Inorganic Biochemistry (2018), 182 (), 230-237CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)
      Copper Transporter 1 (CTR1) is a homotrimeric membrane protein providing the main route of copper transport into eukaryotic cells from the extracellular milieu. Its N-terminal extracellular domain, rich in His and Met residues, is considered responsible for directing copper into the transmembrane channel. Most of vertebrate CTR1 proteins contain the His residue in position three from N-terminus, creating a well-known Amino Terminal Cu(II)- and Ni(II)-Binding (ATCUN) site. CTR1 from humans, primates and many other species contains the Met-Asp-His (MDH) sequence, while some rodents including mouse have the Met-Asn-His (MNH) N-terminal sequence. CTR1 is thought to collect Cu(II) ions from blood copper transport proteins, including albumin, but previous reports indicated that the affinity of N-terminal peptide/domain of CTR1 is significantly lower than that of albumin, casting serious doubt on this aspect of CTR1 function. Using potentiometry and spectroscopic techniques we demonstrated that MDH-amide, a tripeptide model of human CTR1 N-terminus, binds Cu(II) with K of 1.3 × 1013 M-1 at pH 7.4, ∼13 times stronger than Human Serum Albumin (HSA), and MNH-amide is even stronger, K of 3.2 × 1014 M-1 at pH 7.4. These results indicate that the N-terminus of CTR1 may serve as intermediate binding site during Cu(II) transfer from blood copper carriers to the transporter. MDH-amide, but not MNH-amide also forms a low abundance complex with non-ATCUN coordination involving the Met amine, His imidazole and Asp carboxylate. This species might assist Cu(II) relay down the peptide chain or its redn. to Cu(I), both steps necessary for the CTR1 function.
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    65. 65
      Bossak-Ahmad, K.; Mital, M.; Płonka, D.; Drew, S. C.; Bal, W. Oligopeptides Generated by Neprilysin Degradation of β-Amyloid Have the Highest Cu(II) Affinity in the Whole Aβ Family. Inorg. Chem. 2019, 58, 932– 943,  DOI: 10.1021/acs.inorgchem.8b03051
      65
      Oligopeptides generated by neprilysin degradation of β-amyloid have the highest Cu(II) affinity in the whole Aβ Family
      Bossak-Ahmad, Karolina; Mital, Mariusz; Plonka, Dawid; Drew, Simon C.; Bal, Wojciech
      Inorganic Chemistry (2019), 58 (1), 932-943CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
      The catabolism of β-amyloid (Aβ) is carried out by numerous endopeptidases including neprilysin, which hydrolyzes peptide bonds preceding positions 4, 10, and 12 to yield Aβ4-9 and a minor Aβ12-x species. Alternative processing of the amyloid precursor protein by β-secretase also generates the Aβ11-x species. All these peptides contain a Xxx-Yyy-His sequence, also known as an ATCUN or NTS motif, making them strong chelators of Cu(II) ions. We synthesized the corresponding peptides, Phe-Arg-His-Asp-Ser-Gly-OH (Aβ4-9), Glu-Val-His-His-Gln-Lys-am (Aβ11-16), Val-His-His-Gln-Lys-am (Aβ12-16), and pGlu-Val-His-His-Gln-Lys-am (pAβ11-16), and investigated their Cu(II) binding properties using potentiometry, and UV-vis, CD, and ESR spectroscopies. We found that the three peptides with unmodified N-termini formed square-planar Cu(II) complexes at pH 7.4 with analogous geometries but significantly varied Kd values of 6.6 fM (Aβ4-9), 9.5 fM (Aβ12-16), and 1.8 pM (Aβ11-16). Cyclization of the N-terminal Glu11 residue to the pyroglutamate species pAβ11-16 dramatically reduced the affinity (5.8 nM). The Cu(II) affinities of Aβ4-9 and Aβ12-16 are the highest among the Cu(II) complexes of Aβ peptides. Using fluorescence spectroscopy, we demonstrated that the Cu(II) exchange between the Phe-Arg-His and Val-His-His motifs is very slow, on the order of days. These results are discussed in terms of the relevance of Aβ4-9, a major Cu(II) binding Aβ fragment generated by neprilysin, as a possible Cu(II) carrier in the brain.
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    66. 66
      Young, T. R.; Xiao, Z. Principles and practice of determining metal-protein affinities. Biochem. J. 2021, 478, 1085– 1116,  DOI: 10.1042/BCJ20200838
      66
      Principles and practice of determining metal-protein affinities
      Young, Tessa R.; Xiao, Zhiguang
      Biochemical Journal (2021), 478 (5), 1085-1116CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
      A review. Metal ions play many crit. roles in biol., as structural and catalytic cofactors, and as cell regulatory and signalling elements. The metal-protein affinity, expressed conveniently by the metal dissocn. const., KD, describes the thermodn. strength of a metal-protein interaction and is a key parameter that can be used, for example, to understand how proteins may acquire metals in a cell and to identify dynamic elements (e.g. cofactor binding, changing metal availabilities) which regulate protein metalation in vivo. Here, we outline the fundamental principles and practical considerations that are key to the reliable quantification of metal-protein affinities. We review a selection of spectroscopic probes which can be used to det. protein affinities for essential biol. transition metals (including Mn(II), Fe(II), Co(II), Ni(II), Cu(I), Cu(II) and Zn(II)) and, using selected examples, demonstrate how rational probe selection combined with prudent exptl. design can be applied to det. accurate KD values.
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    67. 67
      Sokołowska, M.; Bal, W. Cu(II) complexation by “non-coordinating” N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES buffer). J. Inorg. Biochem. 2005, 99, 1653– 1660,  DOI: 10.1016/j.jinorgbio.2005.05.007
      67
      Cu(II) complexation by "non-coordinating" N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES buffer)
      Sokolowska, Magdalena; Bal, Wojciech
      Journal of Inorganic Biochemistry (2005), 99 (8), 1653-1660CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier B.V.)
      The combined potentiometric and spectroscopic studies of interactions of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) with Cu(II) demonstrated that this popular buffer, commonly labeled as noncoordinating forms a CuL+ complex, with the log βCuL value of 3.22. This complex undergoes alk. hydrolysis above pH 6, resulting in Cu(OH)2 pptn. However, the presence of HEPES at a typical concn. of 100 mM at pH 7.4 elevates the apparent binding const., being detd. for a complex of another ligand, by a factor of 80. HEPES does not form ternary complexes with amino acids Ala, Trp, and His, but may do so with other bioligands, such as nucleotides. Therefore, HEPES can still be recommended for Cu(II) studies in place of other common buffers, such as Tris and phosphate, but appropriate corrections and precautions should be applied in quant. expts.
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    68. 68
      Krężel, A.; Wójcik, J.; Maciejczyk, M.; Bal, W. May GSH and L-His contribute to intracellular binding of zinc? Thermodynamic and solution structural study of a ternary complex. Chem. Commun. 2003, 704– 705,  DOI: 10.1039/b300632h
      68
      May GSH and L-His contribute to intracellular binding of zinc? Thermodynamic and solution structural study of a ternary complex
      Krezel, Artur; Wojcik, Jacek; Maciejczyk, Maciej; Bal, Wojciech
      Chemical Communications (Cambridge, United Kingdom) (2003), (6), 704-705CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      GSH and L-His are abundant biomols. and likely biol. ligands for Zn(II) under certain conditions. Potentiometric titrns. provide evidence of formation of ternary Zn(II) complexes with GSH and L-His or D-His with slight stereoselectivity in favor of L-His (ca. 1 log unit of stability const.). The soln. structure of the ZnH(GSH)(L-His)(H2O) complex at pH 6.8, detd. by NMR, includes tridentate L-His, monodentate (sulfur) GSH, and weak interligand interactions. Calcns. of competitiveness of this complex for Zn(II) binding at pH 7.4 indicate that it is likely to be formed in vivo under conditions of GSH depletion. Otherwise, GSH alone emerges as a likely Zn(I) carrier.
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    69. 69
      Jeżowska-Bojczuk, M.; Kaczmarek, P.; Bal, W.; Kasprzak, K. S. Coordination mode and oxidation susceptibility of nickel(II) complexes with 2’-deoxyguanosine 5′-monophosphate and L-histidine. J. Inorg. Biochem. 2004, 98, 1770– 1777,  DOI: 10.1016/j.jinorgbio.2004.08.002
      69
      Coordination mode and oxidation susceptibility of nickel(II) complexes with 2'-deoxyguanosine 5'-monophosphate and L-histidine
      Jezowska-Bojczuk, Malgorzata; Kaczmarek, Piotr; Bal, Wojciech; Kasprzak, Kazimierz S.
      Journal of Inorganic Biochemistry (2004), 98 (11), 1770-1777CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier B.V.)
      The formation of binary and ternary complexes of Ni(II) with two biol. relevant mols., 2'-deoxyguanosine 5'-monophosphate (dGMP) and L-histidine (histidine or His) was characterized by potentiometry and UV-visible spectroscopy. For dGMP, the mononuclear complexes with stoichiometries NiH2L+, NiHL and NiL- were found. In the mixed system the ternary complexes NiH2LA, NiHLA- and NiLA2- were detected. In binary systems, the Ni(II) ion coordinates to dGMP through the N-7 atom of its purine ring and indirectly through a water mol. bonded to the phosphate group, while in ternary complexes Ni(II) is bonded to all three histidine donors and directly to the phosphate group of dGMP. Both binary and ternary complexes are susceptible to oxidn. by H2O2, with the increased formation of 8-oxo-dGMP in the ternary system. The toxicol. relevance of these findings stems from possible disturbance by the major biol. Ni(II)-His complex of the nucleotide pools homeostasis through the formation of ternary species and oxidn. promotion, as well as from 8-oxo-dGMP capacity to inhibit enzymic elimination of pro-mutagenic oxidized nucleotides from such pools.
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      Sigel, H.; Martin, R. B. Coordinating properties of the amide bond. Stability and structure of metal ion complexes of peptides and related ligands. Chem. Rev. 1982, 82, 385– 426,  DOI: 10.1021/cr00050a003
      70
      Coordinating properties of the amide bond. Stability and structure of metal ion complexes of peptides and related ligands
      Sigel, Helmut; Martin, R. Bruce
      Chemical Reviews (Washington, DC, United States) (1982), 82 (4), 385-426CODEN: CHREAY; ISSN:0009-2665.
      A review with 409 refs.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XlvVars78%253D&md5=2af792cd8dac8d86c46b95077501b160
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      Wilcox, D. E. Isothermal titration calorimetry of metal ions binding to proteins: An overview of recent studies. Inorg. Chim. Acta 2008, 361, 857– 867,  DOI: 10.1016/j.ica.2007.10.032
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      Isothermal titration calorimetry of metal ions binding to proteins: An overview of recent studies
      Wilcox, Dean E.
      Inorganica Chimica Acta (2008), 361 (4), 857-867CODEN: ICHAA3; ISSN:0020-1693. (Elsevier B.V.)
      A review. Isothermal titrn. calorimetry (ITC) is a technique that is capable of quantifying the stoichiometry, equil. consts. and thermodn. of mol. binding events. Thus, important information about the interaction of metal ions with biol. macromols. can be obtained with ITC measurements. This review highlights many of the recent studies of metal ions binding to proteins that have used ITC to quantify the thermodn. of metal-protein interactions.
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      Kostyukevich, Y.; Kononikhin, A.; Popov, I.; Indeykina, M.; Kozin, S. A.; Makarov, A. A.; Nikolaev, E. Supermetallization of peptides and proteins during electrospray ionization. J. Mass Spectrom. 2015, 50, 1079– 1087,  DOI: 10.1002/jms.3622
      72
      Supermetallization of peptides and proteins during electrospray ionization
      Kostyukevich, Yury; Kononikhin, Alexey; Popov, Igor; Indeykina, Maria; Kozin, Sergey A.; Makarov, Alexander A.; Nikolaev, Eugene
      Journal of Mass Spectrometry (2015), 50 (9), 1079-1087CODEN: JMSPFJ; ISSN:1076-5174. (John Wiley & Sons Ltd.)
      The formation of metal-peptide complexes during electrospray ionization (ESI) is a widely known phenomenon and is often considered to be undesirable. Such effect considerably limits the use of ESI mass spectrometry for the study of biol. relevant metal-peptide compds. that are present in the soln. and play crit. roles in many bioprocesses such as progression of neurodegenerative diseases. Under specific conditions such as high temp. of the desolvating capillary, an interesting effect, which can be called supermetallization, occurs. Using a model peptide Aβ amyloid domain 1-16, an increase in the temp. of the desolvating capillary results in multiple substitutions of hydrogen atoms by Zn atoms in this peptide. At high temps. (T ∼ 400°), up to 11 zinc atoms can be covalently bound to (1-16) Aβ. Supermetallization of (1-16) Aβ depends on the solvent compn. and pH. Supermetallization was also demonstrated for proteins, such as ubiquitin and cytochrome C. That proves that the supermetallization is a general phenomenon for peptides and proteins. For the structural study of supermetallized complexes, electron-capture dissocn. (ECD) fragmentation was applied. The effect of hydrogen rearranging during ECD was obsd. In addn., quantum chem. calcns. were used to est. the possible structures of different supermetallized complexes. These results allow a more deep understanding of the limitations of the use of ESI mass spectrometry for the study of biol. relevant metal-peptide complexes.
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      Beech, G. Some recent studies in the thermodynamics of metal complex formation. Q. Rev., Chem. Soc. 1969, 23, 410,  DOI: 10.1039/qr9692300410
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      Vallet, V.; Wahlgren, U.; Grenthe, I. Chelate effect and thermodynamics of metal complex formation in solution: A quantum chemical study. J. Am. Chem. Soc. 2003, 125, 14941– 14950,  DOI: 10.1021/ja036646j
      74
      Chelate Effect and Thermodynamics of Metal Complex Formation in Solution: A Quantum Chemical Study
      Vallet, Valerie; Wahlgren, Ulf; Grenthe, Ingmar
      Journal of the American Chemical Society (2003), 125 (48), 14941-14950CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The accuracy of quantum chem. predictions of structures and thermodn. data for metal complexes depends both on the quantum chem. methods and the chem. models used. A thermodn. analog of the Eigen-Wilkins mechanism for ligand substitution reactions (Model A) turns out to be sufficiently simple to catch the essential chem. of complex formation reactions and allows quantum chem. calcns. at the ab initio level of thermodn. quantities both in gas phase and soln.; the latter by using the conductor-like polarizable continuum (CPCM) model. Model A describes the complex formation as a two-step reaction: [M(H2O)x](aq) + L(aq) .dblharw. [M(H2O)x],L(aq); and [M(H2O)x],L(aq) .dblharw. [M(H2O)x-1L],(H2O)(aq). The first step, the formation of an outer-sphere complex is described using the Fuoss equation and the second, the intramol. exchange between an entering ligand from the second and water in the first coordination shell, using quantum chem. methods. The thermodn. quantities for this model were compared to those for the reaction: [M(H2O)x](aq) + L(aq) .dblharw. [M(H2O)x-1L](aq) + (H2O)(aq) (Model B), as calcd. for each reactant and product sep. The models were tested using complex formation between Zn2+ and ammonia, methylamine, and ethylenediamine, and complex formation and chelate ring closure reactions in binary and ternary UO22+-oxalate systems. The results show that the Gibbs energy of reaction for Model A are not strongly dependent on the no. of water ligands and the structure of the second coordination sphere; it provides a much more precise est. of the thermodn. of complex formation reactions in soln. than that obtained from Model B. The agreement between the exptl. and calcd. data for the formation of Zn(NH3)2+(aq) and Zn(NH3)2+2(aq) is better than 8 kJ/mol for the former, as compared to 30 kJ/mol or larger, for the latter. The Gibbs energy of reaction obtained for the UO2+2 oxalate systems using model B differs between 80 and 130 kJ/mol from the exptl. results, whereas the agreement with Model A is better. The errors in the quantum chem. ests. of the entropy and enthalpy of reaction are somewhat larger than those for the Gibbs energy, but still in fair agreement with expts.; adding water mols. in the second coordination sphere improves the agreement significantly. Reasons for the different performance of the two models are discussed. The quantum chem. data were used to discuss the microscopic basis of exptl. enthalpy and entropy data, to det. the enthalpy and entropy contributions in chelate ring closure reactions and to discuss the origin of the so-called "chelate effect". Contrary to many earlier suggestions, this is not even in the gas phase, a result of changes in translation entropy contributions. There is no simple explanation of the high stability of chelate complexes; it is a result of both enthalpy and entropy contributions that vary from one system to the other.
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jasms.1c00206.

    • Serial dilution data (Figures S1–S6), Cu2+ titrations (Figures S7 and S8) and derived apparent Kd (Figure S9), isotopic distributions for detection of Cu2+ reduction (Figure S10), calculated pH distribution of Cu(II)Aβ4-16 (Figure S11), raw spectra for histidine competition (Figures S12 and S13), literature log β values for Cu(II) complexes of hepc6 and Aβ4-16 (Tables S1 and S2), species distributions for Cu(II) complexes of MNH-NH2, Aβ4-9, Aβ4-16, and MDH-NH2 (Figure S14), stopped-flow spectra (Figure S15), kinetic traces (Figure S16), and recalculation of apparent spray pH (Table S3) (PDF)


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