Temperature-induced reversible conformational change in the first 100 residues of alpha-synuclein

Natively disordered proteins are a growing class of anomalies to the structure-function paradigm. The natively disordered protein alpha-synuclein is the primary component of Lewy bodies, the cellular hallmark of Parkinson's disease. We noticed a dramatic difference in dilute solution 1H-15N Heteronuclear Single Quantum Coherence (HSQC) spectra of wild-type alpha-synuclein and two disease-related mutants (A30P and A53T), with spectra collected at 35 degrees C showing fewer cross-peaks than spectra acquired at 10 degrees C. Here, we show the change to be the result of a reversible conformational exchange linked to an increase in hydrodynamic radius and secondary structure as the temperature is raised. Combined with analytical ultracentrifugation data showing alpha-synuclein to be monomeric at both temperatures, we conclude that the poor quality of the 1H-15N HSQC spectra obtained at 35 degrees C is due to conformational fluctuations that occur on the proton chemical shift time scale. Using a truncated variant of alpha-synuclein, we show the conformational exchange occurs in the first 100 amino acids of the protein. Our data illustrate a key difference between globular and natively disordered proteins. The properties of globular proteins change little with solution conditions until they denature cooperatively, but the properties of natively disordered proteins can vary dramatically with solution conditions.     

Backbone 1H and 15N resonance assignments of the N-terminal SH3 domain of drk in folded and unfolded states using enhanced-sensitivity pulsed field gradient NMR techniques

Summary The backbone ¹H and ¹⁵N resonances of the N-terminal SH3 domain of the Drosophila signaling adapter protein, drk, have been assigned. This domain is in slow exchange on the NMR timescale between folded and predominantly unfolded states. Data were collected on both states simultaneously, on samples of the SH3 in near physiological buffer exhibiting an approximately 1:1 ratio of the two states. NMR methods which exploit the chemical shift dispersion of the ¹⁵N resonances of unfolded states and pulsed field gradient water suppression approaches for avoiding saturation and dephasing of amide protons which rapidly exchange with solvent were utilized for the assignment.Abbreviations 2D, 3D two-, three-dimensional - drkN SH3 N-terminal SH3 domain of Drosophila drk - HSQC heteronuclear single-quantum spectroscopy - NOE nuclear Overhauser enhancement - SH3 Src homology domain 3 - TOCSY total correlation spectroscopy     

Multidimensional NMR identifies the conformational shift essential for catalytic competence in the 60-kDa Drosophila melanogaster dUTPase trimer

The catalytic mechanism of dUTP pyrophosphatase (dUTPase), responsible for the prevention of uracil incorporation into DNA, involves ordering of the flexible C terminus of the enzyme. This conformational shift is investigated by multidimensional NMR on the Drosophila enzyme. Flexible segments of the homotrimer give rise to sharp resonances in the (1)H-(15)N heteronuclear single-quantum coherence (HSQC) spectra, which are clearly distinguishable from the background resonances of the well folded protein globule. Binding of the product dUMP or the analogues dUDP and alpha,beta-imino-dUTP to the enzyme induces a conformational change reflected in the disappearance of eight sharp resonances. This phenomenon is interpreted as nucleotide binding-induced ordering of some residues upon the folded protein globule. Three-dimensional (15)N-edited (1)H-(15)N HSQC total correlation spectroscopy (TOCSY) and (1)H-(15)N HSQC nuclear Overhauser effect spectroscopy measurements allowed clear assignment of these eight specific resonance peaks. The residues identified correspond to the conserved C-terminal sequence motif, indicating that (i) this conformational shift is amenable to NMR studies in solution even in the large trimeric molecule and (ii) formation of the closed enzyme conformer in the case of the Drosophila enzyme does not require the complete triphosphate chain of the substrate. NMR titration of the enzyme with the nucleotide ligands as well as kinetic data indicated significant deviation from the model of independent active sites within the homotrimer. The results suggest allosterism in the eukaryotic dUTPase.     

15N-labeled tRNA. Identification of dihydrouridine in Escherichia coli tRNAfMet, tRNALys, and tRNAPhe by 1H-15N two-dimensional NMR

The N3 imino units of dihydrouridine were identified in samples of 15N-labeled Escherichia coli tRNAfMet, tRNALys, and tRNAPhe by 1H-15N two-dimensional NMR. The peaks for dihydrouridine had high field 1H (9.7-9.8 ppm) and 15N (147.8-149.5 ppm) chemical shifts. Assignments were made by 1H-15N chemical shift correlation based on values obtained in model studies with tri-O-benzoyl- and tri-O-acetyldihydrouridine. The rates of exchange of the imino protons with water suggest that the D-loop in tRNAfMet is less stable than the D-loops in tRNALys or tRNAPhe. Closely spaced peaks were observed for the two dihydrouridines in tRNAPhe in a high resolution spectrum.     

Biophysical characterization of structural properties and folding of interleukin-1 receptor antagonist

Structural properties and folding of interleukin-1 receptor antagonist (IL-1ra), a therapeutically important cytokine with a symmetric beta-trefoil topology, are characterized using optical spectroscopy, high-resolution NMR, and size-exclusion chromatography. Spectral contributions of two tryptophan residues, Trp17 and Trp120, present in the wild-type protein, have been determined from mutational analysis. Trp17 dominates the emission spectrum of IL-1ra, while Trp120 is quenched presumably by the nearby cysteine residues in both folded and unfolded states. The same Trp17 gives rise to two characteristic negative peaks in the aromatic CD. Urea denaturation of the wild-type protein is probed by measuring intrinsic and extrinsic (binding of 1-anilinonaphthalene-8-sulfonic acid) fluorescence, near- and far-UV CD, and 1D and 2D ((1)H-(15)N heteronuclear single quantum coherence (HSQC)) NMR. Overall, the data suggest an essentially two-state equilibrium denaturation mechanism with small, but detectable structural changes within the pretransition region. The majority of the (1)H-(15)N HSQC cross-peaks of the folded state show only a limited chemical shift change as a function of the denaturant concentration. However, the amide cross-peak of Leu31 demonstrates a significant urea dependence that can be fitted to a two-state binding model with a dissociation constant of 0.95+/-0.04 M. This interaction has at least a five times higher affinity than reported values for nonspecific urea binding to denatured proteins and peptides, suggesting that the structural context around Leu31 stabilizes the protein-urea interaction. A possible role of denaturant binding in inducing the pretransition changes in IL-1ra is discussed. Urea unfolding of wild-type IL-1ra is sufficiently slow to enable HPLC separation of folded and unfolded states. Quantitative size-exclusion chromatography has provided a hydrodynamic view of the kinetic denaturation process. Thermodynamic stability and unfolding kinetics of IL-1ra resemble those of structurally and evolutionary close IL-1beta, suggesting similarity of their free energy landscapes.     

Apoflavodoxin (un)folding followed at the residue level by NMR

The denaturant-induced (un)folding of apoflavodoxin from Azotobacter vinelandii has been followed at the residue level by NMR spectroscopy. NH groups of 21 residues of the protein could be followed in a series of 1H-15N heteronuclear single-quantum coherence spectra recorded at increasing concentrations of guanidinium hydrochloride despite the formation of protein aggregate. These NH groups are distributed throughout the whole apoflavodoxin structure. The midpoints of unfolding determined by NMR coincide with the one obtained by fluorescence emission spectroscopy. Both techniques give rise to unfolding curves with transition zones at significantly lower denaturant concentrations than the one obtained by circular dichroism spectroscopy. The NMR (un)folding data support a mechanism for apoflavodoxin folding in which a relatively stable intermediate is involved. Native apoflavodoxin is shown to cooperatively unfold to a molten globule-like state with extremely broadened NMR resonances. This initial unfolding step is slow on the NMR chemical shift timescale. The subsequent unfolding of the molten globule is faster on the NMR chemical shift timescale and the limited appearance of 1H-15N HSQC cross peaks of unfolded apoflavodoxin in the denaturant range studied indicates that it is noncooperative.     

Mapping protein–protein interaction by 13C′-detected heteronuclear NMR spectroscopy

The copper-mediated protein–protein interaction between yeast Atx1 and Ccc2 has been examined by protonless heteronuclear NMR and compared with the already available ¹H–¹⁵N HSQC information. The observed chemical shift variations are analyzed with respect to the actual solution structure, available through intermolecular NOEs. The advantage of using the CON-IPAP spectrum with respect to the ¹H–¹⁵N HSQC resides in the increased number of signals observed, including those of prolines. CBCACO-IPAP experiments allow us to focus on the interaction region and on side-chain carbonyls, while a newly designed CEN-IPAP experiment on side-chains of lysines. An attempt is made to rationalize the chemical shift variations on the basis of the structural data involving the interface between the proteins and the nearby regions. It is here proposed that protonless ¹³C direct-detection NMR is a useful complement to ¹H based NMR spectroscopy for monitoring protein–protein and protein–ligand interactions. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at     

Assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxin

As a necessary first step in the use of heteronuclear correlated spectra to obtain high resolution solution structures of the protein, assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxin (Mr 12,000) uniformly labeled with 15N has been performed. The 15N chemical shifts of backbone amide nitrogen atoms have been determined for both oxidation states of thioredoxin using 15N-1H correlated and two-dimensional heteronuclear single-quantum coherence (HSQC) TOCSY and NOESY spectra. The backbone assignments are complete, except for the proline imide nitrogen resonances and include Gly33, whose amide proton resonance is difficult to observe in homonuclear 1H spectra. The differences in the 15N chemical shift between oxidized and reduced thioredoxin, which occur mainly in the vicinity of the two active site cysteines, including residues distant in the amino acid sequence which form a hydrophobic surface close to the active site, are consistent with the differences observed for proton chemical shifts in earlier work on thioredoxin.     

NMR characterization of interleukin-2 in complexes with the IL-2Ralpha receptor component,and with low molecular weight compounds that inhibit the IL-2/IL-Ralpha interaction

Nuclear magnetic resonance (NMR) methods were employed to study the interaction of the cytokine Interleukin-2 (IL-2) with the alpha-subunit of its receptor (IL-2Ralpha), and to help understand the behavior of small molecule inhibitors of this interaction. Heteronuclear (1)H-(15)N HSQC experiments were used to identify the interaction surface of (15)N-enriched Interleukin-2 ((15)N-IL-2) in complex with human IL-2Ralpha. In these experiments, chemical shift and line width changes in the heteronuclear single-quantum coherence (HSQC) spectra upon binding of (15)N-IL-2 enabled classification of NH atoms as either near to, or far from, the IL-2Ralpha interaction surface. These data were complemented by hydrogen/deuterium (H/D) exchange measurements, which illustrated enhanced protection of slowly-exchanging IL-2 NH protons near the site of interaction with IL-2Ralpha. The interaction surface defined by NMR compared well with the IL-2Ralpha binding site identified previously using mutagenesis of human and murine IL-2. Two low molecular weight inhibitors of the IL-2/IL-2Ralpha interaction were studied: one (a cyclic peptide derivative) was found to mimic a part of the cytokine and bind to IL-2Ralpha; the other (an acylphenylalanine derivative) was found to bind to IL-2. For the interaction between IL-2 and the acylphenylalanine, chemical shift perturbations of (15)N and (15)NH backbone resonances were tracked as a function of ligand concentration. The perturbation pattern observed for this complex revealed that the acylphenylalanine is a competitive inhibitor-it binds to the same site on IL-2 that interacts with IL-2Ralpha.     

The trans labilization of cis-[PtCl2(13CH3NH2)2] by glutathione can be monitored at physiological pH by [1H,13C] HSQC NMR

In order to monitor the trans labilization of cisplatin at physiological pH we have prepared the complex cis-[PtCl₂(¹³CH₃NH₂)₂] and studied its interactions with excess glutathione in aqueous solution at neutral pH by two-dimensional [1H,13C] heteronuclear single-quantum correlation (HSQC) NMR spectroscopy. [1H,13C] HSQC spectroscopy is a good method for following the release of ¹³CH₃NH₂ but is not so good for characterizing the Pt species in solution. In the reaction of cisplatin with glutathione, Pt–S bonds are formed and Pt–NH₃ bonds are broken. The best technique for following the formation of Pt–S bonds of cisplatin is by UV spectroscopy. [1H,13C] HSQC spectroscopy is the best method for following the breaking of the Pt–N bonds. [1H,15N] HSQC spectroscopy is the best method for characterizing the different species in solution. However, the intensity of the peaks in the ¹⁵NH₃–Pt–S region, in [1H,15N] HSQC, reflects a balance between the formation of Pt–S bonds, which increases the signal intensity, and the trans labilization, which decreases the signal intensity. [1H,15N] HSQC spectroscopy and [1H,13C] HSQC spectroscopy are complementary techniques that should be used in conjunction in order to obtain the most accurate information on the interaction of platinum complexes with sulfur-containing ligands.     

Histidine side-chain dynamics and protonation monitored by 13C CPMG NMR relaxation dispersion

The use of ¹³C NMR relaxation dispersion experiments to monitor micro-millisecond fluctuations in the protonation states of histidine residues in proteins is investigated. To illustrate the approach, measurements on three specifically ¹³C labeled histidine residues in plastocyanin (PCu) from Anabaena variabilis (A.v.) are presented. Significant Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion is observed for ¹³C^ε1 nuclei in the histidine imidazole rings of A.v. PCu. The chemical shift changes obtained from the CPMG dispersion data are in good agreement with those obtained from the chemical shift titration experiments, and the CPMG derived exchange rates agree with those obtained previously from ¹⁵N backbone relaxation measurements. Compared to measurements of backbone nuclei, ¹³C^ε1 dispersion provides a more direct method to monitor interchanging protonation states or other kinds of conformational changes of histidine side chains or their environment. Advantages and shortcomings of using the ¹³C^ε1 dispersion experiments in combination with chemical shift titration experiments to obtain information on exchange dynamics of the histidine side chains are discussed.     

Interaction between calcium-free calmodulin and IQ motif of neurogranin studied by nuclear magnetic resonance spectroscopy

The interaction of Ca(2+)-free calmodulin (apoCaM) with the IQ motif corresponding to the calmodulin-binding domain of neurogranin has been studied by nuclear magnetic resonance (NMR) methods. The NMR spectra of uncomplexed apoCaM and apoCaM in complex with the IQ motif recorded at 750 MHz were studied and the backbone assignments of the protein in both forms were obtained by triple-resonance multidimensional NMR experiments. Chemical shift perturbations were used to map the binding surfaces. Only a single set of resonances was observed throughout the titration, indicating that the binding interaction is under fast exchange. Analysis of chemical shift changes indicates that (a) the main interaction and conformational changes occur in the C-terminal domain of calmodulin and (b) linker-1 (residues 40-44) between EF-1 and EF-2, linker-3 (residues 112-117) between EF-3 and EF-4, and the end of the alpha-helix H (residues 145-148) may be involved in the binding process. The dissociation constant (K(d)), estimated by fitting the chemical shift changes against the IQ peptide concentration, ranged from about 1.2 x 10(-5) to 8.8 x 10(-5) M. This result demonstrates that the interaction falls into the weak binding regime.     

A further investigation of the cytochrome<Emphasis Type="Italic"> b</Emphasis><Subscript>5</Subscript>–cytochrome<Emphasis Type="Italic"> c</Emphasis> complex

The interaction of reduced rabbit cytochrome b₅ with reduced yeast iso-1 cytochrome c has been studied through the analysis of ¹H–¹⁵N HSQC spectra, of ¹⁵N longitudinal (R₁) and transverse (R₂) relaxation rates, and of the solvent exchange rates of protein backbone amides. For the first time, the adduct has been investigated also from the cytochrome c side. The analysis of the NMR data was integrated with docking calculations. The result is that cytochrome b₅ has two negative patches capable of interacting with a single positive surface area of cytochrome c. At low protein concentrations and in equimolar mixture, two different 1:1 adducts are formed. At high concentration and/or with excess cytochrome c, a 2:1 adduct is formed. All the species are in fast exchange on the scale of differences in chemical shift. By comparison with literature data, it appears that the structure of one 1:1 adduct changes with the origin or primary sequence of cytochrome b₅.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.Abbreviations HSQC heteronuclear single quantum correlation spectroscopy - MD molecular dynamics     

A preliminary CD and NMR study of the Escherichia coli DNA polymerase III theta subunit.

The theta subunit of DNA polymerase III, the main replicative polymerase of Escherichia coli, has been examined by circular dichroism and by NMR spectroscopy. The polymerase core consists of three subunits: alpha, epsilon, and theta, with alpha possessing the polymerase activity, epsilon functioning as a proofreading exonuclease, and theta, a small subunit of 8.9 kD, of undetermined function. The theta subunit has been expressed in E. coli, and a CD analysis of theta indicates the presence of a significant amount of secondary structure: approximately 52% alpha helix, 9% beta sheet, 21% turns, and 18% random coil. However, at higher concentrations, theta yields a poorly-resolved 1D proton NMR spectrum in which both the amide protons and the methyl protons show poor chemical shift dispersion. Subsequent 1H-15N HSQC analysis of uniformly-15N-labeled theta supports the conclusion that approximately half of the protein is reasonably well-structured. Another quarter of the protein, probably including some of the N-terminal region, is highly mobile, exhibiting a chemical shift pattern indicative of random coil structure. The remaining amide resonances exhibit significant broadening, indicative of intermolecular and/or intramolecular exchange processes. Improved chemical shift dispersion and greater uniformity of resonance intensities in the 1H-15N HSQC spectra resulted when [U-15N]-theta was examined in the presence of epsilon186--the N-terminal domain of the epsilon-subunit. Further work is currently in progress to define the solution structure of theta and the theta-epsilon186 complex.     

Quantitation of rapid proton-deuteron amide exchange using hadamard spectroscopy

The rates of amide proton exchange in protein backbones are very useful reporters of accessibility and structural stability of specific residues and secondary structure elements. Measurement by monitoring changes in intensity of cross-peaks in standard (15)N-(1)H HSQC spectra as protons are replaced by solvent deuterons has become widely accepted. However, these methods are limited to relatively slow rates due to time limitations of the conventional 2D HSQC experiment. Here we show that a Hadamard encoded version of the HSQC, which relies on a multiplexed, frequency selective, excitation in the (15)N dimension, extends application to rates that are as much as an order of magnitude faster than those previously accessible.     

Conformation of membrane-associated proapoptotic tBid

The proapoptotic Bcl-2 family protein Bid is cleaved by caspase-8 to release the C-terminal fragment tBid, which translocates to the outer mitochondrial membrane and induces massive cytochrome c release and cell death. In this study, we have characterized the conformation of tBid in lipid membrane environments, using NMR and CD spectroscopy with lipid micelle and lipid bilayer samples. In micelles, tBid adopts a unique helical conformation, and the solution NMR (1)H/(15)N HSQC spectra have a single well resolved resonance for each of the protein amide sites. In lipid bilayers, tBid associates with the membrane with its helices parallel to the membrane surface and without trans-membrane helix insertion, and the solid-state NMR (1)H/(15)N polarization inversion with spin exchange at the magic angle spectrum has all of the amide resonances centered at (15)N chemical shift (70-90 ppm) and (1)H-(15)N dipolar coupling (0-5 kHz) frequencies associated with NH bonds parallel to the bilayer surface, with no intensity at frequencies associated with NH bonds in trans-membrane helices. Thus, the cytotoxic activity of tBid at mitochondria may be similar to that observed for antibiotic polypeptides, which bind to the surface of bacterial membranes as amphipathic helices and destabilize the bilayer structure, promoting the leakage of cell contents.     

Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H-15N Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1 beta

The application of three-dimensional (3D) heteronuclear NMR spectroscopy to the sequential assignment of the 1H NMR spectra of larger proteins is presented, using uniformly labeled (approximately 95%) [15N]interleukin 1 beta, a protein of 153 residues and molecular mass of 17.4 kDa, as an example. The two-dimensional (2D) 600-MHz spectra of interleukin 1 beta are too complex for complete analysis, owing to extensive cross-peak overlap and chemical shift degeneracy. We show that the combined use of 3D 1H-15N Hartmann-Hahn-multiple quantum coherence (HOHAHA-HMQC) and nuclear Overhauser-multiple quantum coherence (NOESY-HMQC) spectroscopy, designed to provide the necessary through-bond and through-space correlations for sequential assignment, provides a practical general-purpose method for resolving ambiguities which severely limit the analysis of conventional 2D NMR spectra. The absence of overlapping cross-peaks in these 3D spectra allows the unambiguous identification of C alpha H(i)-NH(i+1) and NH(i)-NH(i+1) through-space nuclear Overhauser connectivities necessary for connecting a particular C alpha H(i)-NH(i) through-bond correlation with its associated through-space sequential cross-peak The problem of amide NH chemical shift degeneracy in the 1H NMR spectrum is therefore effectively removed, and the assignment procedure simply involves inspecting a series of 2D 1H-1H slices edited by the chemical shift of the directly bonded 15N atom. Connections between residues can be identified almost without any knowledge of the spin system types involved, though this type of information is clearly required for the eventual placement of the connected residues within the primary sequence.     

A novel way of amino acid-specific assignment in 1H-15N HSQC spectra with a wheat germ cell-free protein synthesis system

For high-throughput protein structural analyses, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as ones utilizing E. coli cells, have been developed, a lot of proteins functioning in solution still were synthesized as insoluble forms. Recently, a novel wheat germ cell-free protein synthesis system was developed, and many of such proteins were synthesized as soluble forms. This means that the applicability of this protein synthesis method to determination of the functional structures of soluble proteins. In our previous work, we synthesized (15)N-labeled proteins with this wheat germ cell-free system, and confirmed this applicability on the basis of the strong similarity between the (1)H-(15)N HSQC spectra for native proteins and the corresponding ones for synthesized ones.In this study, we developed a convenient and reliable method for amino acid selective assignment in (1)H-(15)N HSQC spectra of proteins, using several inhibitors for transaminases and glutamine synthase in the process of protein synthesis. Amino acid selective assignment in (1)H-(15)N HSQC spectra is a powerful means to monitor the features of proteins, such as folding, intermolecular interactions and so on. This is also the first direct experimental evidence of the presence of active transaminases and glutamine synthase in wheat germ extracts.     

NMR studies on the equilibrium unfolding of ketosteroid isomerase by urea

Multidimensional NMR was employed to investigate the structural changes in the urea-induced equilibrium unfolding of the dimeric ketosteroid isomerase (KSI) from Pseudomonas putida biotype B. Sequence specific backbone assignments for the native KSI and the protein with 3.5 M urea were carried out using various 3D NMR experiments. Hydrogen exchange measurements indicated that the secondary structures of KSI were not affected significantly by urea up to 3.5 M. However, the chemical shift analysis of (1)H-(15)N HSQC spectra at various urea concentrations revealed that the residues in the dimeric interface region, particularly around the beta5-strand, were significantly perturbed by urea at low concentrations, while the line-width analysis indicated the possibility of conformational exchange at the interface region around the beta6-strand. The results thus suggest that the interface region primarily around the beta5- and beta6-strands could play an important role as the starting positions in the unfolding process of KSI.     

Redox-related conformational changes in Rhodobacter capsulatus cytochrome c2

WEFT-NOESY and transfer WEFT-NOESY NMR spectra were used to determine the heme proton assignments for Rhodobacter capsulatus ferricytochrome c2. The Fermi contact and pseudo-contact contributions to the paramagnetic effect of the unpaired electron in the oxidized state were evaluated for the heme and ligand protons. The chemical shift assignments for the 1H and 15N NMR spectra were obtained by a combination of 1H-1H and 1H-15N two-dimensional NMR spectroscopy. The short-range nuclear Overhauser effect (NOE) data are consistent with the view that the secondary structure for the oxidized state of this protein closely approximates that of the reduced form, but with redox-related conformational changes between the two redox states. To understand the decrease in stability of the oxidized state of this cytochrome c2 compared to the reduced form, the structural difference between the two redox states were analyzed by the differences in the NOE intensities, pseudo-contact shifts and the hydrogen-deuterium exchange rates of the amide protons. We find that the major difference between redox states, although subtle, involve heme protein interactions, orientation of the heme ligands, differences in hydrogen bond networks and, possible alterations in the position of some internal water molecules. Thus, it appears that the general destabilization of cytochrome c2, which occurs on oxidation, is consistent with the alteration of hydrogen bonds that result in changes in the internal dynamics of the protein.