Hydrogen Exchange Methods Research Articles

Specific non-native hydrophobic interactions in a hidden folding intermediate: implications for protein folding.

Structures of intermediates and transition states in protein folding are usually characterized by amide hydrogen exchange and protein engineering methods and interpreted on the basis of the assumption that they have native-like conformations. We were able to stabilize and determine the high-resolution structure of a partially unfolded intermediate that exists after the rate-limiting step of a four-helix bundle protein, Rd-apocyt b(562), by multidimensional NMR methods. The intermediate has partial native-like secondary structure and backbone topology, consistent with our earlier native state hydrogen exchange results. However, non-native hydrophobic interactions exist throughout the structure. These and other results in the literature suggest that non-native hydrophobic interactions may occur generally in partially folded states. This can alter the interpretation of mutational protein engineering results in terms of native-like side chain interactions. In addition, since the intermediate exists after the rate-limiting step and Rd-apocyt b(562) folds very rapidly (k(f) approximately 10(4) s(-1)), these results suggest that non-native hydrophobic interactions, in the absence of topological misfolding, are repaired too rapidly to slow folding and cause the accumulation of folding intermediates. More generally, these results illustrate an approach for determining the high-resolution structure of folding intermediates.

Thermodynamic and kinetic exploration of the energy landscape of Borrelia burgdorferi OspA by native-state hydrogen exchange.

We report a native-state hydrogen-exchange (HX) method to simultaneously obtain both thermodynamic and kinetic information on the formation of multiple excited states in a folding energy landscape. Our method exploits the inherent dispersion and pH dependence of the intrinsic HX rates to cover both the EX2 (thermodynamic) and EX1 (kinetic) regimes. At each concentration of denaturant, HX measurements are performed over a range of pH values. Using this strategy, we dissected Borrelia burgdorferi OspA, a predominantly beta-sheet protein containing a unique single-layer beta-sheet, into five cooperative units and postulated excited states predominantly responsible for HX. More importantly, we determined the interconversion rates between these excited states and the native state. The use of both thermodynamic and kinetic information from native-state HX enabled us to construct a folding landscape of this 28kDa protein, including local minima and maxima, and to discriminate on-pathway and off-pathway intermediates. This method, which we term EX2/EX1 HX, should be a powerful tool for characterizing the complex folding mechanisms exhibited by the majority of proteins.

Early formation of a beta hairpin during folding of staphylococcal nuclease H124L as detected by pulsed hydrogen exchange.

Pulsed hydrogen exchange methods were used to follow the formation of structure during the refolding of acid-denatured staphylococcal nuclease containing a stabilizing Leu substitution at position 124 (H124L SNase). The protection of more than 60 backbone amide protons in uniformly (15)N-labeled H124L SNase was monitored as a function of refolding time by heteronuclear two-dimensional NMR spectroscopy. As found in previous studies of staphylococcal nuclease, partial protection was observed for a subset of amide protons even at the earliest folding time point (10 msec). Protection indicative of marginally stable hydrogen-bonded structure in an early folding intermediate was observed at over 30 amide positions located primarily in the beta-barrel and to a lesser degree in the alpha-helical domain of H124L SNase. To further characterize the folding intermediate, protection factors for individual amide sites were measured by varying the pH of the labeling pulse at a fixed refolding time of 16 msec. Protection factors >5.0 were observed only for amide positions in a beta-hairpin formed by strands 2 and 3 of the beta-barrel domain and a single site near the C-terminus. The results indicate that formation of stable hydrogen-bonded structure in a core region of the beta-sheet is among the earliest structural events in the folding of SNase and may serve as a nucleation site for further structure formation.

Anabaena apoflavodoxin hydrogen exchange: on the stable exchange core of the alpha/beta(21345) flavodoxin-like family.

An important issue in modern protein biophysics is whether structurally homologous proteins share common stability and/or folding features. Flavodoxin is an archetypal alpha/beta protein organized in three layers: a central beta-sheet (strand order 21345) flanked by helices 1 and 5 on one side and helices 2, 3, and 4 on the opposite side. The backbone internal dynamics of the apoflavodoxin from Anabaena is analyzed here by the hydrogen exchange method. The hydrogen exchange rates indicate that 46 amide protons, distributed throughout the structure of apoflavodoxin, exchange relatively slowly at pH 7.0 (k(ex) < 10(-1) min(-1)). According to their distribution in the structure, protein stability is highest on the beta-sheet, helix 4, and on the layer formed by helices 1 and 5. The exchange kinetics of Anabaena apoflavodoxin was compared with those of the apoflavodoxin from Azotobacter, with which it shares a 48% sequence identity, and with Che Y and cutinase, two other alpha/beta (21345) proteins with no significant sequence homology with flavodoxins. Both similarities and differences are observed in the cores of these proteins. It is of interest that a cluster of a few structurally equivalent residues in the central beta-strands and in helix 5 is common to the cores.

Protein folding intermediates and pathways studied by hydrogen exchange.

In order to solve the immensely difficult protein-folding problem, it will be necessary to characterize the barriers that slow folding and the intermediate structures that promote it. Although protein-folding intermediates are not accessible to the usual structural studies, hydrogen exchange (HX) methods have been able to detect and characterize intermediates in both kinetic and equilibrium modes--as transient kinetic folding intermediates on a subsecond time scale, as labile equilibrium molten globule intermediates under destabilizing conditions, and as infinitesimally populated intermediates in the high free-energy folding landscape under native conditions. Available results consistently indicate that protein-folding landscapes are dominated by a small number of discrete, metastable, native-like partially unfolded forms (PUFs). The PUFs appear to be produced, one from another, by the unfolding and refolding of the protein's intrinsically cooperative secondary structural elements, which can spontaneously create stepwise unfolding and refolding pathways. Kinetic experiments identify three kinds of barrier processes: (a) an initial intrinsic search-nucleation-collapse process that prepares the chain for intermediate formation by pinning it into a condensed coarsely native-like topology; (b) smaller search-dependent barriers that put the secondary structural units into place; and (c) optional error-dependent misfold-reorganization barriers that can cause slow folding, intermediate accumulation, and folding heterogeneity. These conclusions provide a coherent explanation for the grossly disparate folding behavior of different globular proteins in terms of distinct folding pathways.

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

Backbone Dynamics of Triple-helical Collagen-like Structure.

Some details of the backbone dynamics in the collagen-like triple helix is discussed and the role of backbone dynamics in functioning collagen proteins is illustrated. On a series of oligotripeptides synthetic analogs of collagen formation of high-frequency vibrational backbone dynamics and low-frequency nonlinear backbone dynamics upon stepwise elongation of peptide chain have been described using infrared spectroscopy and hydrogen-exchange method. In the fully completed triple helix the level of high-frequency backbone dynamics is regulated firstly by contact interactions of adjacent atoms and chemical bounded groups, while the level of low-frequency large-amplitude backbone dynamics depends mainly on cooperative interactions attributed by conjugation of interpeptide hydrogen bonds. In native collagens the nonlinear large-amplitude dynamics following by non-denaturational micro-unfolding of the triple-helical structure appears to be under the natural selection control delivering an optimal condition for formation, functioning and utilization of collagen fibrils.

Signal transmission between subunits in the hemoglobin T-state

To study allosteric mechanism in hemoglobin, a hydrogen-exchange method was used to measure ligand-dependent changes in structural free energy at defined allosterically sensitive positions. When the two α-subunits are CN-met liganded, effects can be measured locally, within the α-subunit, and also remotely, within the β-subunit, even though the quaternary structure remains in the T conformation. When the two β-subunits are liganded, effects occur at the same positions. The effects seen are the same, independently of whether ligands occupy the α-chain hemes or the β-chain hemes. Control experiments rule out modes of energy transfer other than programmed cross-subunit interaction within the T-state. Cross-subunit transfer may depend on pulling the heme trigger (moving the heme iron into the heme plane) rather than on liganding alone.

Apparent local stability of the secondary structure of Azotobacter vinelandii holoflavodoxin II as probed by hydrogen exchange: implications for redox potential regulation and flavodoxin folding.

As a first step to determine the folding pathway of a protein with an alpha/beta doubly wound topology, the 1H, 13C, and 15N backbone chemical shifts of Azotobacter vinelandii holoflavodoxin II (179 residues) have been determined using multidimensional NMR spectroscopy. Its secondary structure is shown to contain a five-stranded parallel beta-sheet (beta2-beta1-beta3-beta4-beta5) and five alpha-helices. Exchange rates for the individual amide protons of holoflavodoxin were determined using the hydrogen exchange method. The amide protons of 65 residues distributed throughout the structure of holoflavodoxin exchange slowly at pH* 6.2 [kex < 10(-5) s(-1)] and can be used as probes in future folding studies. Measured exchange rates relate to apparent local free energies for transient opening. We propose that the amide protons in the core of holoflavodoxin only exchange by global unfolding of the apo state of the protein. The results obtained are discussed with respect to their implications for flavodoxin folding and for modulation of the flavin redox potential by the apoprotein. We do not find any evidence that A. vinelandii holoflavodoxin II is divided into two subdomains based on its amide proton exchange rates, as opposed to what is found for the structurally but not sequentially homologous alpha/beta doubly wound protein Che Y.

Stability and solvent accessibility of SecA protein of Escherichia coli.

It has been shown that many proteins, when converted into partially unfolded structures, interact strongly with a lipid bilayer. SecA protein of Escherichia coli is an unusual water-soluble protein which, in the native form, can readily penetrate the membrane and lipid bilayer. In order to see whether the native SecA exhibits partially unfolded characteristics, the stability and solvent accessibility of SecA were studied using various spectroscopic and hydrogen-exchange methods. The results are compared with the reported data for native and molten globule forms of alpha-lactalbumin (alpha-LA), as well as those for apocytochrome c. The exposure of hydrophobic residues of SecA, as monitored by 8-anilinonaphthalene-1-sulfonic acid (ANS) binding, and the extent of amide hydrogen exchange were comparable to those of native alpha-LA. On the other hand, equilibrium unfolding experiments showed that SecA is less stable than native alpha-LA. The results of tryptic digestion and the change of endogenous ATPase activity induced by guanidine hydrochloride were suggestive of the C-terminal half of SecA being more flexible than the rest of the protein. The overall conclusion is that the SecA as a whole has a somewhat open structure due to a relatively unstable C-domain which may facilitate its penetration into a lipid bilayer.

Stability and dynamics in a hyperthermophilic protein with melting temperature close to 200 degrees C.

The rubredoxin protein from the hyperthermophilic archaebacterium Pyrococcus furiosus was examined by a hydrogen exchange method. Even though the protein does not exhibit reversible thermal unfolding, one can determine its stability parameters-free energy, enthalpy, entropy, and melting temperature-and also the distribution of stability throughout the protein, by using hydrogen exchange to measure the reversible cycling of the protein between native and unfolded states that occurs even under native conditions.

Amide hydrogen exchange and internal dynamics the chemotactic protein CheY from Escherichia coli

The backbone internal dynamics of the wild-type 129 amino acid α/β parallel protein CheY and its double mutant F14N/P110G are analysed here by the hydrogen-exchange method. The F14N mutation is known to stabilise the protein and to accelerate refolding while P110G is destabilising and accelerates unfolding. We first assigned and characterised the double mutant by nuclear magnetic resonance (NMR), to try and discover any possible conformational change induced by the two mutations. The main difference between the two proteins is a favourable N-capping interaction of the newly introduced Asn14 side-chain at the beginning of the first α-helix (α-helix A). Second, we have measured the exchange rates in the wild-type and mutant CheY. In the first case the observed protection factors are slightly dispersed around an average value. According to their distribution in the structure, protein stability is highest on one face of the central β-sheet, in the surroundings of the main hydrophobic core formed by side-chains of residues in β-strands I, II and III and helices A and E. The mutations in the double mutant protein affect two distinct subdomains differently (from β-strand I to III and from α-helix C to the end). In the second subdomain the number of protected protons is reduced with respect to those in the wild-type. This differential behaviour can be explained by a selective decrease in stability of the second folding subdomain produced by the P110G mutation and the opposite effect in the first subdomain, produced by the F14N mutation. α-Helix A, which is involved together with β-strands I and III in the folding nucleus of CheY, shows the largest protection factors in both proteins.

Hydrogen exchange: the modern legacy of Linderstrøm-Lang.

This discussion, prepared for the Protein Society's symposium honoring the 100th anniversary of Kaj Linderstrøm-Lang, shows how hydrogen exchange approaches initially conceived and implemented by Lang and his colleagues some 50 years ago are contributing to current progress in structural biology. Examples are chosen from the active protein folding field. Hydrogen exchange methods now make it possible to define the structure of protein folding intermediates in various contexts: as tenuous molten globule forms at equilibrium under destabilizing conditions, in kinetic intermediates that exist for less than one second, and as infinitesimally populated excited state forms under native conditions. More generally, similar methods now find broad application in studies of protein structure, energetics, and interactions. This article considers the rise of these capabilities from their inception at the Carlsberg Labs to their contemporary role as a significant tool of modern structural biology.

Future directions in folding: the multi-state nature of protein structure.

All possible protein folding intermediates exist in equilibrium with the native protein at native as well as non-native conditions, with occupation determined by their free energy level. The study of these forms can illuminate the fundamental principles of protein structure and folding. Hydrogen exchange methods can be used to detect and characterize these partially unfolded forms at native conditions and as a function of mild denaturant and temperature. This information illuminates the requirements that govern the ability of kinetic and equilibrium methods to study folding intermediates.

Rapid amide proton exchange rates in peptides and proteins measured by solvent quenching and two-dimensional NMR.

In an effort to develop a more versatile quenched hydrogen exchange method for studies of peptide conformation and protein-ligand interactions, the mechanism of amide proton exchange for model peptides in DMSO-D2O mixtures was investigated by NMR methods. As in water, H-D exchange rates in the presence of 90% or 95% DMSO exhibit characteristic acid- and base-catalyzed processes and negligible water catalysis. However, the base-catalyzed rate is suppressed by as much as four orders of magnitude in 95% DMSO. As a result, the pH at which the exchange rate goes through a minimum is shifted up by about two pH units and the minimum exchange rate is approximately 100-fold reduced relative to that in D2O. The solvent-dependent decrease in base-catalyzed exchange rates can be attributed primarily to a large increase in pKa values for the NH group, whereas solvent effects on pKW seem less important. Addition of toluene and cyclohexane resulted in improved proton NMR chemical shift dispersion. The dramatic reduction in exchange rates observed in the solvent mixture at optimal pH makes it possible to apply 2D NMR for NH exchange measurements on peptides under conditions where rates are too rapid for direct NMR analysis. To test this solvent-quenching method, melittin was exchanged in D2O (pH 3.2, 12 degrees C), aliquots were quenched by rapid freezing, lyophilized, and dissolved in quenching buffer (70% DMSO, 25% toluene, 4% D2O, 1% cyclohexane, 75 mM dichloroacetic acid) for NMR analysis. Exchange rates for 21 amide protons were measured by recording 2D NMR spectra on a series of samples quenched at different times. The results are consistent with a monomeric unfolded conformation of melittin at acidic pH. The ability to trap labile protons by solvent quenching makes it possible to extend amide protection studies to peptide ligands or labile protons on the surface of a protein involved in macromolecular interactions.

Hydrogen exchange rates and protein folding

Transient folding intermediates have been monitored in 11 proteins by pulsed hydrogen exchange methods. Standard hydrogen isotope exchange kinetics have also been measured for several molten globules. Taken together with folding kinetics detected by optical techniques, and with hydrogen exchange kinetics in native proteins, the results permit conclusions about time constants, structure and stability of folding intermediates, and about parallel versus serial folding pathways.

Backbone dynamics of (Pro-Hyp-Gly)10 and a designed collagen-like triple-helical peptide by 15N NMR relaxation and hydrogen-exchange measurements.

The backbone dynamics of specific residues in two collagen-like triple-helical peptides with (X-Y-Gly)n sequences have been investigated using two-dimensional inverse-detected 15N NMR relaxation measurements and hydrogen-exchange experiments. One peptide, (POG)10, has the highest possible imino acid content and is considered to be a very stable prototype of a triple helix. The second peptide, (POG)3ITGARGLAGPOG(POG)3 (denoted T3-785), models an imino acid poor region of type III collagen and contains 12 residues from near the unique collagenase cleavage site. 15N relaxation parameters and hydrogen-exchange data were obtained for a glycine residue in the center of (POG)10 and for the tripeptide unit Gly-Leu-Ala in the middle of T3-785. Analysis of the relaxation data of the rodlike triple-helical peptides required the assumption of anisotropic overall motion, and the model-free approach of Lipari and Szabo (1982) was used to derive overall motional parameters and the order parameter, S2, that describes the amplitudes of the internal motion. First the mobilities of the Gly, Leu, and Ala residues in peptide T3-785 were compared. Both hydrogen-exchange methods and relaxation measurements indicated that the residue in the Y position (Ala) is more mobile than residues in the Gly and X positions (Leu). The slower exchange rates of Gly and Leu compared to that of Ala are consistent with the two-hydrogen-bonded model for the triple helix. Then the backbone mobilities of the central Gly residue were compared for the two peptides (POG)10 and T3-785. In this case, 15N relaxation measurements give different results from hydrogen exchange. The glycine residues in the trimer form of both T3-785 and (POG)10 have high values for the order parameter (near 0.85), suggesting similar small-amplitude internal motions and rigid backbones in both peptides. In contrast to the similar values of the order parameters, hydrogen-exchange data indicate that the central Gly exchanges at a faster rate in the trimer form of T3-785 than in (POG)10. These results suggest that a Gly in the imino acid rich environment of (POG)10 is dynamically different from a Gly in the imino acid poor environment of T3-785 and that the difference lies in the slower motion related to stability, rather than the faster motion on the picosecond time scale. This sequence-dependent difference in dynamical properties may have important consequences for recognition processes in collagen.

Structure and stability of the molten globule state of guinea-pig alpha-lactalbumin: a hydrogen exchange study.

A partially folded state of guinea pig alpha-lactalbumin (the A-state or molten globule state), formed by denaturation at low pH, has been studied using hydrogen exchange methods. The overall distribution of exchange kinetics, measured by 1-D NMR, suggests that fewer than 20 amides in the structure are involved in highly persistent residual structure, although CD results suggest that many other parts of the chain are folded, for a significant proportion of the time, into less stable structural elements. The pH-jump experiments show that some amides that are strongly protected from exchange in the native state become freely accessible in the A-state but that conversely a majority, at least, of those that are slow to exchange in the A-state retain that protection in the native state. This suggests that the persistent structure in the A-state is native-like although the possibility that nonnative like structural elements persist cannot be eliminated. Resonance assignments for key residues in the NMR spectrum of the native state have enabled us to use the pH-jump method also to identify the majority of the most protected amides in the A-state: they are located in two hydrophobic segments, corresponding to the B- and C-helices of the native structure. This strongly suggests that the most persistent structure of the A-state includes these regions. A variety of lines of evidence, including fluorescence quenching data and, most remarkably, very effective protection from exchange of an indole NH in a tryptophan side chain, suggest that some form of hydrophobic core in the helical domain of the native structure persists in the A-state, although without the stereochemical rigidity of the native tertiary structure. The other domain of the native structure, including the beta-sheet, appears not to contain structural elements which persist to the same extent in the A-state, emphasizing that the molten globule is highly heterogeneous, in terms of the stability and specificity of both backbone and side chain interactions.

Helix-coil theories: a comparative study for finite length polypeptides

At present, numerous polypeptides of known sequence and length are being synthesized by chemical or biosynthetic means. The observations of the helix-coil transition is one of the important methods of studying the factors which stabilize {alpha}-helices of short to moderate length in these molecules. As a result, the observation and interpretation of helix-coil transitions is likely to return to its prominence of 25 years ago, but with increased power because of the precise sequence information and the ability to observe the state of individual amino acid residues or peptide groups by nuclear magnetic resonance spectroscopy and hydrogen-exchange methods. In this paper we develop some of the details of the helix-coil model, which are likely to be useful in the interpretation of these more incisive experiments. 37 refs., 2 figs.