Native-state Hydrogen Exchange Research Articles

Towards a consistent modeling of protein thermodynamic and kinetic cooperativity: how applicable is the transition state picture to folding and unfolding?

To what extent do general features of folding/unfolding kinetics of small globular proteins follow from their thermodynamic properties? To address this question, we investigate a new simplifed protein chain model that embodies a cooperative interplay between local conformational preferences and hydrophobic burial. The present four-helix-bundle 55mer model exhibits protein-like calorimetric two-state cooperativity. It rationalizes native-state hydrogen exchange observations. Our analysis indicates that a coherent, self-consistent physical account of both the thermodynamic and kinetic properties of the model leads naturally to the concept of a native state ensemble that encompasses considerable conformational fluctuations. Such a multiple-conformation native state is seen to involve conformational states similar to those revealed by native-state hydrogen exchange. Many of these conformational states are predicted to lie below native baselines commonly used in interpreting calorimetric data. Folding and unfolding kinetics are studied under a range of intrachain interaction strengths as in experimental chevron plots. Kinetically determined transition midpoints match well with their thermodynamic counterparts. Kinetic relaxations are found to be essentially single-exponential over an extended range of model interaction strengths. This includes the entire unfolding regime and a significant part of a folding regime with a chevron rollover, as has been observed for real proteins that fold with non-two-state kinetics. The transition state picture of protein folding and unfolding is evaluated by comparing thermodynamic free energy profiles with actual kinetic rates. These analyses suggest that some chevron rollovers may arise from an internal frictional effect that increasingly impedes chain motions with more native conditions, rather than being caused by discrete deadtime folding intermediates or shifts of the transition state peak as previously posited.

Lack of definable nucleation sites in the rate-limiting transition state of barnase under native conditions

It has been shown that the burst-phase (submillisecond) intermediate of barnase, if it exists, can be only marginally more stable than the fully unfolded state at pH 6.3 and 25 °C. In the study reported here, no stable burst-phase intermediate could be detected, even in the presence of stabilizing salt (0.4 M Na2SO4). These results suggest that a burst-phase intermediate with even marginal stability does not exist. The absence of such an intermediate in turn suggests the need for re-examination of the rate-limiting transition state (RLTS) under native conditions, which was previously characterized by using a three-state model with a stable intermediate and protein engineering. Surprisingly, mutations throughout the structure of barnase do not significantly affect the folding rate, suggesting a lack of specific favorable interactions among the side-chains in the RLTS. This RLTS is clearly different from that previously characterized under denaturing conditions, indicating that changes take place in the RLTS under native and denaturing conditions. The occurrence of such changes is further supported by the observation that the unfolding rate constants of barnase and its mutants were divergent or convergent as a function of denaturant concentrations. Consistent with changes in the RLTS, a re-analysis of data from native-state hydrogen exchange studies has shown that the logarithm of the unfolding rate constant inflects down under low concentrations of denaturant. Here, we discuss in detail the question of whether changes in the RLTS involve a kinetically silent intermediate that occurs after the initial RLTS.

Native-state energetics of a thermostabilized variant of ribonuclease HI

Escherichia coli RNase HI is a well-characterized model system for protein folding and stability. Controlling protein stability is critical for both natural proteins and for the development of engineered proteins that function under extreme conditions. We have used native-state hydrogen exchange on a variant containing the stabilizing mutation Asp10 to alanine in order to determine its residue-specific stabilities. On average, the ΔGunf value for each residue was increased by 2-3 kcal/mol, resulting in a lower relative population of partially unfolded forms. Though increased in stability by a uniform factor, D10A shows a distribution of stabilities in its secondary structural units that is similar to that of E. coli RNase H, but not the closely related protein from Thermus thermophilus. Hence, the simple mutation used to stabilize the enzyme does not recreate the balance of conformational flexibility evolved in the thermophilic protein.

A kinetic folding intermediate probed by native state hydrogen exchange

Stopped-flow fluorescence studies on the N-terminal domain of rat CD2 (CD2.d1) have demonstrated that folding from the fully denatured state (U) proceeds via the transient accumulation of an apparent intermediate (I) in a so-called burst phase that precedes the rate-limiting transition leading to the native state (N). A previous pH-dependent equilibrium hydrogen exchange (HX) study identified a subset of amides in CD2.d1 which, under EX2 conditions, exchange from N with free energies greater than or equal to the free energy difference between the N and I states calculated from the stopped-flow data. Under EX1 conditions the rates of HX for these amides tend towards an asymptote that matches the global unfolding rate calculated from the stopped-flow data, suggesting that exchange for these amides requires traversing the N-to-I transition state barrier. Exchange for these amides presumably occurs from exchange-competent forms comprising the kinetic burst phase therefore. To explore this idea further, native state HX (NHX) data have been collected for CD2.d1 under EX2 conditions using denaturant concentrations which span either side of the denaturant concentration where, according to the stopped-flow data, the apparent U and I states are iso-energetic. The data fit to a two-component, sub-global (sg)/global (g) NHX mechanism, yielding ΔG and m value parameters (where the m value is a measure of hydrocarbon solvation). Regression analysis demonstrates that the (msg, ΔGsg) and (mg, ΔGg) values calculated for this subset of amides correspond with those describing the kinetic burst phase transition. This result confirms the ability of the NHX technique to explore the structural and energetic properties of kinetic folding intermediates.

Ensemble modulation as an origin of denaturant-independent hydrogen exchange in proteins

Native state hydrogen exchange (HX) has become a powerful tool for the analysis of conformational states that exist under native conditions. However, the interpretation of HX data in terms of conformational fluctuations is still controversial. In particular, it has been shown that many residues display exchange behavior that is independent of denaturant concentration. It has been postulated that this lack of denaturant dependence results from local fluctuations that do not expose appreciable amounts of buried surface area. Here, we use a general thermodynamic description of HX to explore the different possibilities for this behavior. We find that the denaturant dependence seen in HX experiments under native conditions is not a de facto indication of the amount of surface area exposure required for exchange. Instead, this behavior results from the relatively homogenous character of the conformational ensemble that exists under native conditions and the non-specific nature of denaturant effects. Furthermore, a comparison of the HX behavior from a stabilized mutant of Staphylococcal nuclease (SNase) with that predicted for the wild-type SNase from the COREX algorithm suggests that denaturant-independent exchange of many residues is consistent with significant (∼10 %) surface area exposure for this protein.

A statistical appraisal of native state hydrogen exchange data: evidence for a burst phase continuum?

For a number of proteins, folding occurs via the rapid accumulation of secondary and tertiary structural features in a so-called burst phase, preceding the relatively slow, highly activated transition leading to the native state. A fundamental question is: do these burst phase reactions comprise two phase-separated thermodynamic states or a continuum of states? Ribonuclease HI (RNase H) from Escherichia coli and phage T4 lysozyme (T4L) both exhibit such a phenomenon. Native-state hydrogen exchange (NHX) data have been collected for these proteins, providing residue-specific free energies and m-values (a measure of hydrocarbon solvation) for the manifold of partially unfolded, exchange-competent forms that are accessible from the native state (ΔGsg and msg, where the sg subscript denotes sub-global). There is good evidence that these parameters pertain to exchange-competent species comprising the burst phase observed in the global folding kinetics. We combine the results from the global folding kinetics of these proteins with a statistical analysis of their NHX parameters to determine if the distribution of experimental (msg, ΔGsg) values derive from a mechanism where the burst phase is two-state. For RNase H, this analysis demonstrates that the burst phase of this protein is not two-state; the results imply a distribution of states, m and ΔG exhibiting a linear functional relationship consistent with the global folding parameters. For T4L, it is difficult to distinguish the observed distribution of msg, ΔGsg values from that expected for a mechanism where the burst phase is two-state. The results for RNase H∗ lend support for the idea that the burst phase reaction of this protein comprises a continuum of states. This has important implications for how we model the process of structural acquisition in protein folding reactions.

Microscopic stability of cold shock protein A examined by NMR native state hydrogen exchange as a function of urea and trimethylamine N-oxide.

Native state hydrogen exchange of cold shock protein A (CspA) has been characterized as a function of the denaturant urea and of the stabilizing agent trimethylamine N-oxide (TMAO). The structure of CspA has five strands of beta-sheet. Strands beta1-beta4 have strongly protected amide protons that, based on experiments as a function of urea, exchange through a simple all-or-none global unfolding mechanism. By contrast, the protection of amide protons from strand beta5 is too weak to measure in water. Strand beta5 is hydrogen bonded to strands beta3 and beta4, both of which afford strong protection from solvent exchange. Gaussian network model (GNM) simulations, which assume that the degree of protection depends on tertiary contact density in the native structure, accurately predict the strong protection observed in strands beta1-beta4 but fail to account for the weak protection in strand beta5. The most conspicuous feature of strand beta5 is its low sequence hydrophobicity. In the presence of TMAO, there is an increase in the protection of strands beta1-beta4, and protection extends to amide protons in more hydrophilic segments of the protein, including strand beta5 and the loops connecting the beta-strands. TMAO stabilizes proteins by raising the free energy of the denatured state, due to highly unfavorable interactions between TMAO and the exposed peptide backbone. As such, the stabilizing effects of TMAO are expected to be relatively independent of sequence hydrophobicity. The present results suggest that the magnitude of solvent exchange protection depends more on solvent accessibility in the ensemble of exchange susceptible conformations than on the strength of hydrogen-bonding interactions in the native structure.

Structural distribution of stability in a thermophilic enzyme

Stability parameters for individual residues in Thermus thermophilus cysteine-free RNase H were determined by native state hydrogen exchange, thus providing a unique comparison of regional thermodynamics between thermophilic and mesophilic homologues. The general distribution of stability in the thermophilic protein is similar to that of its mesophilic homologue, with a proportional increase in stability for almost all residues. As a consequence, the residue-specific stabilities of the two proteins are remarkably similar under conditions where their global stabilities are the same. These results indicate that T. thermophilus RNase H is stabilized in a delocalized fashion, preserving a finely tuned balance of stabilizing interactions throughout the structure. Therefore, although protein stability can be altered by single amino acid substitution, evolution for optimal function may require more subtle and delocalized mechanisms.

The cooperativity of burst phase reactions explored

The denaturant-dependence of the major, observable relaxation rates for folding (kobs) of ribonuclease HI from Escherichia coli (RNase H) and phage T4 lysozyme (T4L) reveal that, for both proteins, folding begins with the rapid and transient accumulation of intermediate species in a “burst phase” which precedes the rate-limiting formation of the native state; this is evidenced by a “rollover” in the folding limb of the rate profiles (kobsversus denaturant, or chevron plot). These rate profiles are most simply described by a three-state mechanism (unfolded-to-intermediate-to-native), which implies that the burst phase represents a transition between two distinct thermodynamic states. It is shown here that the equilibrium properties of these burst phase reactions can be equally well modeled by a mechanism involving a continuum of states where the free energy of each state is linearly related to its m-value (the parameter describing the linear relationship between free energy and denaturant). A numerical model is also developed to describe the time evolution of such a system, which exhibits nearly perfect exponential behavior. Both models emphasize how a continuum of states operating under a linear free energy relationship may behave like a two state system. Such a scheme finds experimental justification from an interpretation of recent native state hydrogen exchange data. The analytical model described for a continuum can account for the observed kinetic profiles of several other model proteins. The results, however, appear context specific, suggesting that burst phase reactions are not entirely random and non-specific. The results reported in this study have important implications for the concept of cooperativity in protein folding reactions.

Relationship between the native-state hydrogen exchange and the folding pathways of barnase.

The previous native-state hydrogen exchange experiment with barnase failed to detect any partially unfolded intermediate state which was contrary to the experimental results from kinetic deuterium hydrogen exchange pulse labeling and protein engineering studies. This has been taken to suggest that the native-state hydrogen exchange method cannot be used alone as an analytical tool to study the folding pathways of proteins. Here, we revisited the pulse labeling experiment with barnase and detected no stable folding intermediate. This finding allows a reconciliation of the native-state HX data and the folding pathway of barnase. Along with alternative theoretical interpretations for a curved chevron plot of protein folding, these data suggest that further investigation of the nature of the intermediate of barnase is needed.

Review of the fourth Johns Hopkins Protein Folding Meeting

Review of the fourth Johns Hopkins Protein Folding Meeting

NMR Hydrogen Exchange of the OB-fold Protein LysN as a Function of Denaturant: The Most Conserved Elements of Structure are the Most Stable to Unfolding

The structure of LysN contains an OB-fold motif composed of a structurally conserved five-stranded β-barrel capped by a poorly conserved α-helix between strands β3 and β4. Two additional α-helices, unique to the LysN structure, flank the N terminus of the OB-fold. The stability of LysN to unfolding has been investigated with NMR native state hydrogen exchange measurements as a function of guanidinium hydrochloride concentration, and equilibrium unfolding transitions monitored by ellipticity at 222nm and fluorescence at 350nm. The spectrophotometric measurements suggest an apparent two-state unfolding transition with ΔGu(0)∼6kcal/mol andm ∼3kcal/(molM). By contrast, NMR hydrogen exchange measurements manifest a distribution of ΔGu(0) and m values which indicate that the protein can undergo subglobal unfolding. The largest ΔGu(0) values from hydrogen exchange are for residues in the β-sheet of the protein. These values, which reflect complete unfolding of the protein, are between 3 and 4kcal/mol higher than those obtained from circular dichroism or fluorescence. This discrepancy may be due to the comparison of NMR hydrogen exchange parameters measured at residue-level resolution, with spectrophotometric parameters that reflect an unresolved super position of unfolding transitions of the α-helices and β-strands. The largest ΔGu(0) values obtained from hydrogen exchange for the subset of residues in the α-helices of the protein, agree with the ΔGu(0) values obtained from circular dichroism or fluorescence. Based on the hydrogen exchange data, however, the three α-helices of LysN are on average 3kcal/mol less stable than the β-sheet. Consistent with the subglobal unfolding of LysN evinced by hydrogen exchange, a deletion mutant that lacks the first α-helix of the protein retains a cooperatively folded structure. Taken together with previous results on the OB-fold proteins SN and CspA, the present results for LysN suggest that the most conserved elements of structure in the OB-fold motif are the most resistant to denaturation. In all three proteins, stability to denaturation correlates with sequence hydrophobicity.

Characterization of internal motions ofEscherichia coli ribonuclease H by Monte Carlo simulation

The backbone dynamics of Escherichia coli ribonuclease H (RNase H) is studied by a recently developed off-lattice Monte Carlo/Metropolis simulation technique. A low-resolution model (virtual-bond model) is used together with knowledge-based potentials. The calculated mean-square fluctuations in alpha carbons are in good agreement with crystallographic temperature factors. The conformations generated around the native state are analyzed by time-dependent orientational and conformational correlation functions to study the internal motions of RNase at different time windows. A correlation between the free-energy changes for native-state hydrogen exchange (HX) and the extent of the autocorrelation in the rotations of the virtual bonds at long times has been observed. Cross-correlations between the rotations of the bonds, which are near-neighbor in the sequence, are effective in all time windows and help the secondary structures to preserve their kinetic stability. On the other hand, the existence of cross-correlations at long times help the tertiary contacts be maintained. The order parameter of NH bond vector for each residue has been calculated and compared with 15N-NMR relaxation measurements.

Characterization of internal motions of Escherichia coli ribonuclease H by Monte Carlo simulation

The backbone dynamics of Escherichia coli ribonuclease H (RNase H) is studied by a recently developed off-lattice Monte Carlo /Metropolis simulation technique. A low-resolution model (virtual-bond model) is used together with knowledge-based potentials. The calculated mean-square fluctuations in alpha carbons are in good agreement with crystallographic temperature factors. The conformations generated around the native state are analyzed by time-dependent orientational and conformational correlation functions to study the internal motions of RNase at different time windows. A correlation between the free-energy changes for native-state hydrogen exchange (HX) and the extent of the autocorrelation in the rotations of the virtual bonds at long times has been observed. Cross-correlations between the rotations of the bonds, which are near-neighbor in the sequence, are effective in all time windows and help the secondary structures to preserve their kinetic stability. On the other hand, the existence of cross-correlations at long times help the tertiary contacts be maintained. The order parameter of NH bond vector for each residue has been calculated and compared with 15N-NMR relaxation measurements. Proteins 1999;34:533–539. © 1999 Wiley-Liss, Inc.

Evidence for an unfolding and refolding pathway in cytochrome c.

It has been suggested that three partially unfolded forms detected in a native state hydrogen exchange study of oxidized cytochrome c may represent sequential intermediates in an unfolding-refolding pathway. To better define the structure of each intermediate a 'stability labeling' method was used in which the stability of a given segment against unfolding was changed. The condition--folded or unfolded--of the stability-labeled segment in each intermediate could then be determined. The structures found are discrete and native-like and are just those necessary for a sequential pathway.

Molten globule unfolding monitored by hydrogen exchange in urea.

The molten globule state of Escherichia coli ribonuclease H1 was studied by hydrogen exchange in order to understand how the energetics of individual regions react to the presence of denaturant. Hydrogen exchange rates were monitored (1) directly by NMR spectroscopy and (2) indirectly by quenching the exchange process and returning to the native state for NMR detection. Direct hydrogen exchange on the molten globule state demonstrated that the observed protons exchange 3-100-fold more slowly than in an unfolded peptide. The quenched hydrogen exchange experiments were modeled after the recently developed "native state hydrogen exchange" experiment and were carried out as a function of urea concentration. The free energy of hydrogen exchange varied linearly with denaturant concentration for all 29 measurable protons, suggesting that each amide's exchange behavior can be modeled with only one type of opening transition. The free energy of unfolding measured by hydrogen exchange and corresponding m values varied for each residue implying a noncooperative molten globule structure. These results are in contrast to similar exchange experiments on native proteins which generally display more than one type of exchange behavior. The single type of exchange seen in the molten globule is probably due to its larger conformational freedom and noncooperative nature.

Microsecond protein folding kinetics from native-state hydrogen exchange.

Native-state amide proton (NH) exchange in turkey ovomucoid third domain (OMTKY3) has been used to determine rates of unfolding and folding at the 13 most slowly exchanging residues. Ten of the 13 NHs have previously been demonstrated to exchange via complete unfolding of OMTKY3 while the remaining three exchange more slowly than expected on the basis of thermal stability alone [Swint-Kruse, L., Robertson, A. D. (1996) Biochemistry 35, 171-180]. Rates of unfolding and folding have been determined by monitoring MH exchange over a range of pH where (1) the free energy of unfolding for third domain, about 7 kcal/mol, is insensitive to pH and (2) the mechanism of exchange changes from one governed by a rapid equilibrium preceding the chemistry of exchange (i.e., EX2 exchange) to one where exchange is limited by the rate of unfolding (i.e., EX1 exchange). The pH dependence of exchange has then been fit to a two-state model to obtain the unfolding and folding rates. Unfolding rates at these 13 NHs in native third domain range from 0.003 to >/= 0.03 s-1. No correlation is observed between opening rates and the free energies measured at the same NHs: for example, the slowest and most rapid opening rates occur at Leu 23 and Asn 33, respectively, and these two NHs show very similar free energies of 6.7 and 6.9 kcal/mol, respectively. In contrast, folding rates show a positive correlation (R2 = 0.90) with free energies, the most rapid folding occurring at the sites with the largest free energies. folding rates are most rapid, 10(3)-10(4) s-1, in the middle of the helix, intermediate rates of around 10(3) s-1 are found in the remainder of the helix and through much of the beta-sheet, and the slowest folding, 10(2)-10(3) s-1, occurs at the juncture between the helix and sheet. Overall, MH exchange from native proteins provides remarkable structural and temporal precision for measuring very rapid conformational fluctuations.

Protein folding intermediates: native-state hydrogen exchange.

The hydrogen exchange behavior of native cytochrome c in low concentrations of denaturant reveals a sequence of metastable, partially unfolded forms that occupy free energy levels reaching up to the fully unfolded state. The step from one form to another is accomplished by the unfolding of one or more cooperative units of structure. The cooperative units are entire omega loops or mutually stabilizing pairs of whole helices and loops. The partially unfolded forms detected by hydrogen exchange appear to represent the major intermediates in the reversible, dynamic unfolding reactions that occur even at native conditions and thus may define the major pathway for cytochrome c folding.

Stable submolecular folding units in a non-compact form of cytochrome c

Studies of structure, dynamics, and stability of cytochrome c (cyt c) at low pH in a non-compact pre-molten globule state indicate that the protein contains submolecular folding units that are independently stable. In high salt, acid cyt c (pD 2·3; where D is deuterium) is nearly as compact as the native form. Nuclear magnetic resonance (n.m.r.) line broadening typical of the molten globule form is seen, indicating loosened packing and increased mobility not only for side-chains but also for the main chain. As NaCl concentration is decreased below 0·05 m, cyt c expands due to the deshielding of electrostatic repulsions, attaining a linear extent perhaps double that of the native protein (viscosity, fluorescence). In the extended form, tertiary structural hydrogen bonds are largely broken (hydrogen exchange rate), some normally buried parts of the protein are exposed to water (fluorescence), and many of the native side-chain contacts must be lost. Nevertheless, almost all of the helical content is retained (circular dichroism). The helices involve the same amino acid residues that are helical in the native state (hydrogen exchange labeling monitored by 2-dimensional n.m.r.). The equilibrium constant for helix formation at 20°C (0·02 m-NaCl, pD 2·2) is about 10 (hydrogen exchange rate), even though the individual helical segments when isolated have little or no structure. Additional experiments were done to check assumptions and calibrate parameters that underlie the hydrogen exchange analysis of protein folding. These results indicate that the native-like helical segments in the expanded non-globular form of cyt c exist as part of somewhat larger submolecular folding units that possess significant equilibrium stability. Results from equilibrium and kinetic studies of protein folding support the generality of this conclusion. This view is contrary to the two-state paradigm for equilibrium folding and inconsistent with the idea that side-chain packing constraints determine folding motifs. The result suggests an extension of the thermodynamic hypothesis for protein structure to kinetic folding processes, so that the amino acid code for equilibrium and kinetic folding may be the same, and also seems pertinent to the biological evolution of contemporary protein structures.