Characterization of transient protein folding intermediates during myoglobin reconstitution by time-resolved electrospray mass spectrometry with on-line isotopic pulse labeling

A novel technique for studying protein folding kinetics is presented. It is based on a continuous-flow setup that is coupled to an electrospray (ESI) mass spectrometer and allows initiation of a folding reaction, followed by isotopic pulse labeling. The protein is electrosprayed "quasi-instantaneously" after exposure to the deuterated solvent. This approach yields structural information from the ESI charge state distribution and from the H/D exchange levels of individual protein states, while at the same time noncovalent interactions can be monitored. This technique is used to study the reconstitution of holomyoglobin (hMb) from unfolded apomyoglobin (aMb) and free heme. MS/MS is used to establish that a short-lived folding intermediate with two heme groups attached represents a protein-bound heme dimer. This state appears to have a compactness close to that of native hMb; however, isotopic labeling indicates a significantly perturbed structure. Another intermediate is bound to a single heme group and shows a charge state distribution similar to that of unfolded aMb. Exchange levels exhibited by this state are lower than for unfolded aMb, indicating that fewer hydrogens are exposed to the solvent and/or that more of them are involved in hydrogen bonding. Native hMb leads to the formation of low charge state ions (hMb(9+), hMb(8+)) and shows low exchange levels. However, early during reconstitution, a slightly unfolded form of the heme-protein complex contributes to the observed hMb(9+) ions. A peak width analysis reveals that the structural heterogeneity of some of the observed protein species decreases as reconstitution proceeds.     

Structural and dynamic characteristics of a partially folded state of ubiquitin revealed by hydrogen exchange mass spectrometry

Structural and dynamic properties of a partially folded conformation (A-state) of ubiquitin are studied using amide hydrogen exchange in solution (HDX) and mass spectrometric detection. A clear distinction between the native state of the protein and the A-state can be made when HDX is carried out in a semicorrelated regime. Convoluted exchange patterns are interpreted with the aid of HDX simulations in a three-state system (highly structured, partially unstructured, and fully unstructured states). The data clearly indicate a highly dynamic character of the non-native state. Furthermore, combination of HDX and protein ion fragmentation in the gas phase [by means of collision-induced dissociation (CAD)] is used to evaluate the conformational stability of various protein segments specifically in the molten globular state. Chain flexibility appears to be distributed very unevenly in this non-native conformation. The highest degree of structural disorder is displayed by the C-terminal segment (Gly(53)-Gly(76)), which was previously suggested to form a transient alpha-helix. The least dynamic segment of ubiquitin in the A-state is Thr(9)-Glu(18) (which was previously suggested to form a stable nativelike beta-strand), with the adjacent segments exhibiting somewhat diminished conformational stability. The study also demonstrates the power of mass spectrometry as a tool in providing conformer-specific information about the structure and dynamics of both native and non-native protein states coexisting in solution under equilibrium.     

Methanol-induced conformations of myoglobin at pH 4.0

The equilibrium methanol-induced conformation changes of holomyoglobin (hMb) at pH 4.0 have been studied by circular dichroism, tryptophan fluorescence, and Soret band absorption and by electrospray ionization mass spectrometry (ESI-MS). Optical spectra show the following: (1) In 35-40% (v/v) methanol/water, the native-like secondary structure remains, the tertiary structure is lost, the heme protein interactions are decreased, and a folding intermediate is formed. (2) In 50% methanol, heme is lost from the protein, and there is a small decrease in helicity together with a loss of tertiary structure. (3) At >60% methanol, the helicity increases and the apoprotein goes into a helical denatured state. The conformations are also probed by the charge states produced in ESI-MS and by hydrogen/deuterium (H/D) exchange with mass measurement by ESI-MS. At 0-30% methanol, native hMb produces relatively low charge states (9(+)-13(+)) in ESI-MS and exchanges relatively few hydrogens. In 35-40% methanol, at which an intermediate is formed, there is a bimodal distribution of hMb ions with both low (9(+)-13(+)) and high (14(+)-23(+)) charge states and also a high charge state distribution (12(+)-26(+)) of apomyoglobin (aMb) ions. Low and high charge states of hMb and a high charge state of aMb all show the same H/D exchange rate, indicating that an unfolded hMb intermediate interconverts between folded hMb and unfolded aMb. The charge state distribution for the unfolded hMb intermediate observed here is similar to that of the recently reported transient intermediate formed during the acid denaturation of hMb. At 50% alcohol the protein produces predominantly high charge states of aMb ions and shows H/D exchange rates close to those of the acid-denatured protein. H/D exchange of the helical denatured protein at alcohol concentrations >60%, at which high charge states of aMb are produced, shows that the protein structure is more protected than at approximately 50% methanol.     

Mapping protein energy landscapes with amide hydrogen exchange and mass spectrometry: I. A generalized model for a two-state protein and comparison with experiment

Protein amide hydrogen exchange (HDX) is a convoluted process, whose kinetics is determined by both dynamics of the protein and the intrinsic exchange rate of labile hydrogen atoms fully exposed to solvent. Both processes are influenced by a variety of intrinsic and extrinsic factors. A mathematical formalism initially developed to rationalize exchange kinetics of individual amide hydrogen atoms is now often used to interpret global exchange kinetics (e.g., as measured in HDX MS experiments). One particularly important advantage of HDX MS is direct visualization of various protein states by observing distinct protein ion populations with different levels of isotope labeling under conditions favoring correlated exchange (the so-called EX1 exchange mechanism). However, mildly denaturing conditions often lead to a situation where the overall HDX kinetics cannot be clearly classified as either EX1 or EX2. The goal of this work is to develop a framework for a generalized exchange model that takes into account multiple processes leading to amide hydrogen exchange, and does not require that the exchange proceed strictly via EX1 or EX2 kinetics. To achieve this goal, we use a probabilistic approach that assigns a transition probability and a residual protection to each equilibrium state of the protein. When applied to a small protein chymotrypsin inhibitor 2, the algorithm allows complex HDX patterns observed experimentally to be modeled with remarkably good fidelity. On the basis of the model we are now in a position to begin to extract quantitative dynamic information from convoluted exchange kinetics.     

Protein structural dynamics at the gas/water interface examined by hydrogen exchange mass spectrometry

Gas/water interfaces (such as air bubbles or foam) are detrimental to the stability of proteins, often causing aggregation. This represents a potential problem for industrial processes, for example, the production and handling of protein drugs. Proteins possess surfactant-like properties, resulting in a high affinity for gas/water interfaces. The tendency of previously buried nonpolar residues to maximize contact with the gas phase can cause significant structural distortion. Most earlier studies in this area employed spectroscopic tools that could only provide limited information. Here we use hydrogen/deuterium exchange (HDX) mass spectrometry (MS) for probing the conformational dynamics of the model protein myoglobin (Mb) in the presence of N₂ bubbles. HDX/MS relies on the principle that unfolded and/or highly dynamic regions undergo faster deuteration than tightly folded segments. In bubble-free solution Mb displays EX2 behavior, reflecting the occurrence of short-lived excursions to partially unfolded conformers. A dramatically different behavior is seen in the presence of N₂ bubbles; EX2 dynamics still take place, but in addition the protein shows EX1 behavior. The latter results from interconversion of the native state with conformers that are globally unfolded and long-lived. These unfolded species likely correspond to Mb that is adsorbed to the surface of gas bubbles. N₂ sparging also induces aggregation. To explain the observed behavior we propose a simple model, that is, “semi-unfolded” ↔ “native” ↔ “globally unfolded” → “aggregated”. This model quantitatively reproduces the experimentally observed kinetics. To the best of our knowledge, the current study marks the first exploration of surface denaturation phenomena by HDX/MS.     

Ligand binding to heme proteins: connection between dynamics and function

Ligand binding to heme proteins is studied by using flash photolysis over wide ranges in time (100 ns-1 ks) and temperature (10-320 K). Below about 200 K in 75% glycerol/water solvent, ligand rebinding occurs from the heme pocket and is nonexponential in time. The kinetics is explained by a distribution, g(H), of the enthalpic barrier of height H between the pocket and the bound state. Above 170 K rebinding slows markedly. Previously we interpreted the slowing as a "matrix process" resulting from the ligand entering the protein matrix before rebinding. Experiments on band III, an inhomogeneously broadened charge-transfer band near 760 nm (approximately 13,000 cm-1) in the photolyzed state (Mb*) of (carbonmonoxy)myoglobin (MbCO), force us to reinterpret the data. Kinetic hole-burning measurements on band III in Mb* establish a relation between the position of a homogeneous component of band III and the barrier H. Since band III is red-shifted by 116 cm-1 in Mb* compared with Mb, the relation implies that the barrier in relaxed Mb is 12 kJ/mol higher than in Mb*. The slowing of the rebinding kinetics above 170 K hence is caused by the relaxation Mb*----Mb, as suggested by Agmon and Hopfield [(1983) J. Chem. Phys. 79, 2042-2053]. This conclusion is supported by a fit to the rebinding data between 160 and 290 K which indicates that the entire distribution g(H) shifts. Above about 200 K, equilibrium fluctuations among conformational substates open pathways for the ligands through the protein matrix and also narrow the rate distribution. The protein relaxations and fluctuations are nonexponential in time and non-Arrhenius in temperature, suggesting a collective nature for these protein motions. The relaxation Mb*----Mb is essentially independent of the solvent viscosity, implying that this motion involves internal parts of the protein. The protein fluctuations responsible for the opening of the pathways, however, depend strongly on the solvent viscosity, suggesting that a large part of the protein participates. While the detailed studies concern MbCO, similar data have been obtained for MbO2 and CO binding to the beta chains of human hemoglobin and hemoglobin Zürich. The results show that protein dynamics is essential for protein function and that the association coefficient for binding from the solvent at physiological temperatures in all these heme proteins is governed by the barrier at the heme.     

Heme pocket disorder in myoglobin: reversal by acid-induced soft refolding

The protein folding process of heme proteins entails generation of not only a correct global polypeptide structure, but also a correct, functionally competent heme environment. We employed a variety of spectroscopic approaches to probe the structure and dynamics of the heme pocket of a recombinant sperm whale myoglobin. The conformational characteristics were examined by circular dichroism, time-resolved fluorescence spectroscopy, FTIR spectroscopy, and optical absorption spectroscopy in the temperature range 300-20 K. Each of these spectroscopic probes detected modifications confined exclusively to the heme pocket of the expressed myoglobin relative to the native protein. The functional properties were examined by measuring the kinetics of CO binding after flash-photolysis. The kinetics of the expressed myoglobin were more heterogeneous than those of the native protein. Mild acid exposure of the ferric derivative of the recombinant protein resulted in a protein with "nativelike" spectroscopic properties and homogeneous CO binding kinetics. The heme pocket modifications observed in this recombinant myoglobin do not derive from inverted heme. In contrast, when native apomyoglobin is reconstituted with the heme in vitro, the heme pocket disorder could be attributed exclusively to 180 degrees rotation of the bound heme [La Mar, G. N., Toi, H., and Krishnamoorthi, R. (1984) J. Am. Chem. Soc. 106, 6395-6401; Light, W. R., Rohlfs, R. J., Palmer, G., and Olson, J. S. (1987) J. Biol. Chem. 262, 46-52]. We conclude that exposure to low pH decreases the affinity of globin for the heme and allows an extended conformational sampling or "soft refolding" to a nativelike conformation.     

Regulatory interactions between ubiquinol oxidation and ubiquinone reduction sites in the dimeric cytochrome bc1 complex

We have obtained evidence for conformational communication between ubiquinol oxidation (center P) and ubiquinone reduction (center N) sites of the yeast bc1 complex dimer by analyzing antimycin binding and heme bH reduction at center N in the presence of different center P inhibitors. When stigmatellin was occupying center P, concentration-dependent binding of antimycin occurred only to half of the center N sites. The remaining half of the bc1 complex bound antimycin with a slower rate that was independent of inhibitor concentration, indicating that a slow conformational change needed to occur before half of the enzyme could bind antimycin. In contrast, under conditions where the Rieske protein was not fixed proximal to heme bL at center P, all center N sites bound antimycin with fast and concentration-dependent kinetics. Additionally, the extent of fast cytochrome b reduction by menaquinol through center N in the presence of stigmatellin was approximately half of that observed when myxothiazol was bound at center P. The reduction kinetics of the bH heme by decylubiquinol in the presence of stigmatellin or myxothiazol were also consistent with a model in which fixation of the Rieske protein close to heme bL in both monomers allows rapid binding of ligands only to one center N. Decylubiquinol at high concentrations was able to abolish the biphasic binding of antimycin in the presence of stigmatellin but did not slow down antimycin binding rates. These results are discussed in terms of half-of-the-sites activity of the dimeric bc1 complex.     

Discriminating changes in protein structure using tyrosine conjugation

Chemical modification of proteins has been crucial in engineering protein‐based therapies, targeted biopharmaceutics, molecular probes, and biomaterials. Here, we explore the use of a conjugation‐based approach to sense alternative conformational states in proteins. Tyrosine has both hydrophobic and hydrophilic qualities, thus allowing it to be positioned at protein surfaces, or binding interfaces, or to be buried within a protein. Tyrosine can be conjugated with 4‐phenyl‐3H‐1,2,4‐triazole‐3,5(4H)‐dione (PTAD). We hypothesized that individual protein conformations could be distinguished by labeling tyrosine residues in the protein with PTAD. We conjugated tyrosine residues in a well‐folded protein, bovine serum albumin (BSA), and quantified labeled tyrosine with liquid chromatography with tandem mass spectrometry. We applied this approach to alternative conformations of BSA produced in the presence of urea. The amount of PTAD labeling was found to relate to the depth of each tyrosine relative to the protein surface. This study demonstrates a new use of tyrosine conjugation using PTAD as an analytic tool able to distinguish the conformational states of a protein.     

Circular dichroic investigation of the native and non-native conformational states of the growth factor receptor-binding protein 2 N-terminal src homology domain 3: effect of binding to a proline-rich peptide from guanine nucleotide exchange factor

SH3 (src homology domain 3) domains are small protein modules that interact with proline-rich peptides. The structure of the N-terminal SH3 domain from growth factor receptor-binding protein 2 (Grb2), an adapter protein in the intracellular signaling pathway to Ras, was investigated by circular dichroic (CD) spectroscopy. The compact native beta-barrel conformation, previously elucidated by NMR spectroscopy, was largely predominant at pH = 4.8, in the absence of salt. From the structural changes induced by varying pH, ionic strength, temperature, or hydrophobicity of the environment, evidence for the existence of distinct nonnative conformations was obtained in the far- and near-UV domains. Along the free energy scale, these appear to distribute into two conformational ensembles, depending on the extent of structural and thermodynamic differences compared to the native conformation. The first ensemble consists of non-native conformations with a nativelike secondary structure, and the second is composed of partially unfolded conformations having short alpha-helical fragments or turnlike motifs in their nonnative secondary structure. Most of the observed nonnative conformations exist in mild or nondenaturing conditions. They probably have distinct compactness of their inner structure, depending on the strength of nonlocal interactions, but only the native all-beta conformation possesses a condensed protein exterior, appropriate for the binding to the VPPPVPPRRR decapeptide from Sos. Upon binding, the native conformation undergoes a local tertiary structure change in a hydrophobic pocket at the binding site. This is accompanied by the PP-II helix folding of the proline-rich peptide. Interestingly, in the near-UV domain, a significant change in the spectral contribution of an aromatic exciton was observed, thus allowing quantitative tracking of the binding process.     

Assessment of differences in the conformational flexibility of hepatitis B virus core‐antigen and e‐antigen by hydrogen deuterium exchange‐mass spectrometry

Hepatitis B virus core-antigen (capsid protein) and e-antigen (an immune regulator) have almost complete sequence identity, yet the dimeric proteins (termed Cp149_d and Cp(−10)149_d, respectively) adopt quite distinct quaternary structures. Here we use hydrogen deuterium exchange-mass spectrometry (HDX-MS) to study their structural properties. We detect many regions that differ substantially in their HDX dynamics. Significantly, whilst all regions in Cp(−10)149_d exchange by EX2-type kinetics, a number of regions in Cp149_d were shown to exhibit a mixture of EX2- and EX1-type kinetics, hinting at conformational heterogeneity in these regions. Comparison of the HDX of the free Cp149_d with that in assembled capsids (Cp149_c) indicated increased resistance to exchange at the C-terminus where the inter-dimer contacts occur. Furthermore, evidence of mixed exchange kinetics were not observed in Cp149_c, implying a reduction in flexibility upon capsid formation. Cp(−10)149_d undergoes a drastic structural change when the intermolecular disulphide bridge is reduced, adopting a Cp149_d-like structure, as evidenced by the detected HDX dynamics being more consistent with Cp149_d in many, albeit not all, regions. These results demonstrate the highly dynamic nature of these similar proteins. To probe the effect of these structural differences on the resulting antigenicity, we investigated binding of the antibody fragment (Fab E1) that is known to bind a conformational epitope on the four-helix bundle. Whilst Fab E1 binds to Cp149_c and Cp149_d, it does not bind non-reduced and reduced Cp(−10)149_d, despite unhindered access to the epitope. These results imply a remarkable sensitivity of this epitope to its structural context.     

Enzyme conformational dynamics during catalysis and in the 'resting state' monitored by hydrogen/deuterium exchange mass spectrometry

This work reports the use of electrospray mass spectrometry for studying the conformational dynamics of enzymes by amide hydrogen/deuterium exchange (HDX) measurements. A rapid-mixing quench-flow approach allows comparisons to be made between the HDX kinetics of free enzymes with those under steady-state conditions. Experiments carried out on carboxypeptidase B in the absence of substrate and in the presence of saturating concentrations of hippuryl-Arg result in HDX kinetics that are indistinguishable. This finding implies that the conformational dynamics that mediate HDX are not significantly different in the resting state of the enzyme and during substrate turnover.     

Synthesis and characterization of a beta-hairpin peptide that represents a 'core module' of bovine pancreatic trypsin inhibitor (BPTI)

A new strategy for the design and construction of peptide fragments that can achieve defined, nativelike secondary structure is presented. The strategy is based upon the hypothesis that 'core elements' of a protein, synthesized in a single polypeptide molecule, will favor nativelike structure, and that by incorporating a cross-link, nativelike core structure will dominate the ensemble as the more extended conformations are excluded. 'Core elements' are the elements of packed secondary structure that contain the slowest exchanging backbone amide protons in the native protein. The 'core elements' in bovine pancreatic trypsin inhibitor (BPTI) are the two long strands of antiparallel beta-sheet (residues 18-24 and 29-35) and the small beta-bridge (residues 43-44). To test the design strategy, we synthesized an 'oxidized core module', which contains the antiparallel strands connected by a modified reverse turn (A27 replaced by D), a natural disulfide cross-link at the open end of the hairpin, and N- and C-termini blocking groups. A peptide with identical sequence but lacking the disulfide cross-link at the open end was used as the 'reduced core module' control. The conformational behavior of both peptides was examined using (1)H NMR spectroscopy. Chemical shift dispersion, chemical shift deviation from random coil values, sequential and long-range NOEs, and H/D amide exchange rates were compared for the two peptides. We conclude that the ensemble of oxidized and reduced core module conformations samples both nativelike 4:4 and non-native 3:5 beta-hairpin structure, and that the oxidized module samples nativelike structure for a greater fraction of the time than the reduced module.     

Coupling between conformation and proton binding in proteins

Interest centers here on whether the use of a fixed charge distribution of a protein solute, or a treatment that considers proton-binding equilibria by solving the Poisson equation, is a better approach to discriminate native from non-native conformations of proteins. In this analysis of the charge distribution of 7 proteins, we estimate the solvation free energy contribution to the total free energy by exploring the 2(zeta) possible ionization states of the whole molecule, with zeta being the number of ionizable groups in the amino acid sequence, for every conformation in the ensembles of 7 proteins. As an additional consideration of the role of electrostatic interactions in determining the charge distribution of native folds, we carried out a comparison of alternative charge assignment models for the ionizable residues in a set of 21 native-like proteins. The results of this work indicate that (1) for 6 out of 7 proteins, estimation of solvent polarization based on the Generalized Born model with a fixed charge distribution provides the optimal trade-off between accuracy, with respect to the Poisson equation, and speed when compared to the accessible surface area model; for the seventh protein, consideration of all possible ionization states of the whole molecule appears to be crucial to discriminate the native from non-native conformations; (2) significant differences in the degree of ionization and hence the charge distribution for native folds are found between the different charge models examined; (3) the stability of the native state is determined by a delicate balance of all the energy components, and (4) conformational entropy, and hence the dynamics of folding, may play a crucial role for a successful ab initio protein folding prediction.     

Crystal structures of myoglobin-ligand complexes at near-atomic resolution.

We have used x-ray crystallography to determine the structures of sperm whale myoglobin (Mb) in four different ligation states (unligated, ferric aquomet, oxygenated, and carbonmonoxygenated) to a resolution of better than 1.2 A. Data collection and analysis were performed in as much the same way as possible to reduce model bias in differences between structures. The structural differences among the ligation states are much smaller than previously estimated, with differences of <0.25 A root-mean-square deviation among all atoms. One structural parameter previously thought to vary among the ligation states, the proximal histidine (His-93) azimuthal angle, is nearly identical in all the ferrous complexes, although the tilt of the proximal histidine is different in the unligated form. There are significant differences, however, in the heme geometry, in the position of the heme in the pocket, and in the distal histidine (His-64) conformations. In the CO complex the majority conformation of ligand is at an angle of 18 +/- 3 degrees with respect to the heme plane, with a geometry similar to that seen in encumbered model compounds; this angle is significantly smaller than reported previously by crystallographic studies on monoclinic Mb crystals, but still significantly larger than observed by photoselection. The distal histidine in unligated Mb and in the dioxygenated complex is best described as having two conformations. Two similar conformations are observed in MbCO, in addition to another conformation that has been seen previously in low-pH structures where His-64 is doubly protonated. We suggest that these conformations of the distal histidine correspond to the different conformational substates of MbCO and MbO(2) seen in vibrational spectra. Full-matrix refinement provides uncertainty estimates of important structural parameters. Anisotropic refinement yields information about correlated disorder of atoms; we find that the proximal (F) helix and heme move approximately as rigid bodies, but that the distal (E) helix does not.     

Unfolding studies of tissue transglutaminase

Activation of tissue transglutaminase by calcium involves a conformational change which allows exposition of the active site to the substrate via movements of domains 3 and 4 that lead to an increase of the inter-domain distance. The inhibitor GTP counteracts these changes. Here we investigate the possible existence of non-native conformational states still compatible with the enzyme activity produced by chemical and thermal perturbations. The results indicate that chemical denaturation is reversible at low guanidine concentrations but irreversible at high concentrations of guanidine. Indeed, at low guanidine concentrations tissue TG-ase exists in a non-native state which is still affected by the ligands as in the native form. In contrast, thermal unfolding is always irreversible, with aggregation and protein self-crosslinkage in the presence of calcium. DSC thermograms of the native protein in the absence of ligands consist of two partly overlapped transitions, which weaken in the presence of calcium and merge together and strengthen in the presence of GTP. Overall, the present work shows, for the first time, the reversible denaturation of a TG-ase isoenzyme and suggests the possibility that also in in vivo, the enzyme may acquire non-native conformations relevant to its patho-physiological functions.     

On the nature of conformational openings: native and unfolded-state hydrogen and thiol-disulfide exchange studies of ferric aquomyoglobin.

Native-state amide hydrogen exchange (HX) of proteins in the presence of denaturant has provided valuable details on the structures of equilibrium folding intermediates. Here, we extend HX theory to model thiol group exchange (SX) in single cysteine-containing variants of sperm whale ferric aquomyoglobin. SX is complementary to HX in that it monitors conformational opening events that expose side-chains, rather than the main chain, to solvent. A simple two-process model, consisting of EX2-limited local structural fluctuations and EX1-limited global unfolding, adequately accounts for all HX data. SX is described by the same model except at very low denaturant concentrations and when the bulky labeling reagent 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) is used. Under these conditions SX can occur by a novel denaturant-dependent process. This anomalous behavior is not observed when the smaller labeling reagent methyl methanethiosulfonate is employed, suggesting that it reflects a denaturant-induced increase in the amplitudes of local structural fluctuations. It also is not seen in heme-free apomyoglobin, which may indicate that local openings are sufficiently large in the absence of denaturant to allow DTNB unhindered access. Differences in SX kinetics obtained using the two labeling reagents provide estimates of the sizes of local opening reactions at different sites in the protein. At all sequence positions examined except for position 73, the same opening event appears to facilitate exchange of both backbone amide and side-chain thiol groups. The C73 thiol group is exposed by a low-energy fluctuation that does not expose its amide group to exchange.     

Water penetration and binding to ferric myoglobin

Flash photolysis investigations of horse heart metmyoglobin bound with NO (Mb(3+)NO) reveal the kinetics of water entry and binding to the heme iron. Photodissociation of NO leaves the sample in the dehydrated Mb(3+) (5-coordinate) state. After NO photolysis and escape, a water molecule enters the heme pocket and binds to the heme iron, forming the 6-coordinate aquometMb state (Mb(3+)H2O). At longer times, NO displaces the H2O ligand to reestablish equilibrium. At 293 K, we determine a value k(w) approximately 5.7 x 10(6) s(-1) for the rate of H2O binding and estimate the H2O dissociation constant as 60 mM. The Arrhenius barrier height H(w) = 42 +/- 3 kJ/mol determined for H2O binding is identical to the barrier for CO escape after photolysis of Mb(2+)CO, within experimental uncertainty, consistent with a common mechanism for entry and exit of small molecules from the heme pocket. We propose that both processes are gated by displacement of His-64 from the heme pocket. We also observe that the bimolecular NO rebinding rate is enhanced by 3 orders of magnitude both for the H64L mutant, which does not bind water, and for the H64G mutant, where the bound water is no longer stabilized by hydrogen bonding with His-64. These results emphasize the importance of the hydrogen bond in stabilizing H2O binding and thus preventing NO scavenging by ferric heme proteins at physiological NO concentrations.     

Ligand-induced monomerization of <Emphasis Type="Italic">Allochromatium vinosum</Emphasis> cytochrome <Emphasis Type="Italic">c</Emphasis>′ studied using native mass spectrometry and fluorescence resonance energy transfer

Cytochrome c' from Allochromatium vinosum is an attractive model protein to study ligand-induced conformational changes. This homodimeric protein dissociates into monomers upon binding of NO, CO or CN(-) to the iron of its covalently attached heme group. While ligand binding to the heme has been well characterized using a variety of spectroscopic techniques, direct monitoring of the subsequent monomerization has not been reported previously. Here we have explored two biophysical techniques to simultaneously monitor ligand binding and monomerization. Native mass spectrometry allowed the detection of the dimeric and monomeric forms of cytochrome c' and even showed the presence of a CO-bound monomer. The kinetics of the ligand-induced monomerization were found to be significantly enhanced in the gas phase compared with the kinetics in solution, however. Ligand binding to the heme and the dissociation of the dimer in solution were also studied using energy transfer from a fluorescent probe to both heme groups of the protein. Comparison of ligand binding kinetics as observed with UV-vis spectroscopy with changes in fluorescence suggested that binding of one CO molecule per dimer could be sufficient for monomerization.     

Pulsed hydrogen exchange and electrospray charge-state distribution as complementary probes of protein structure in kinetic experiments: implications for ubiquitin folding

The possible involvement of "hidden" kinetic intermediates in the apparent two-state folding of some proteins is currently a matter of debate. This study uses time-resolved electrospray ionization (ESI) mass spectrometry with on-line pulsed hydrogen-deuterium exchange (HDX) for monitoring the refolding of acid/methanol-denatured ubiquitin. It is demonstrated that the ESI charge-state distribution (CSD) and the extent of HDX represent nonredundant probes of the protein structure in solution. When considered in isolation, the data provided by both of these probes are consistent with a two-state behavior, involving only denatured ubiquitin D and refolded protein F. However, a careful comparison of the CSD and HDX kinetics reveals the presence of an additional species, exhibiting a CSD like the folded protein but showing non-native HDX characteristics. This kinetic intermediate, D*, is in rapid equilibrium with D, such that the overall reaction is consistent with the mechanism D <--> D* --> F. The results of this work suggest that the occurrence of transient intermediates may be more widespread than commonly thought, especially in cases where a cursory analysis indicates two-state behavior.