Protein Hydrogen Exchange in Denaturant: Quantitative Analysis by a Two-Process Model.

Qian, Hong; Mayo, Stephen L.; Morton, Andrew

doi:10.1021/bi00193a001

Cited by 58 publications

(56 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For these hydrogens the curve for ΔG op against GdmCl concentration extrapolates to the value for global ΔG u at zero GdmCl concentration and, at high GdmCl concentration, merges smoothly with ΔG u values obtained from equilibrium melting data (11). Results for ribonuclease A (15,16) show similar agreement (11). Thus the exchange of the slowest hydrogens in these proteins is dominated by and can measure the parameters of the global unfolding equilibrium (Eqs.…”

Section: Global Local and Subglobal Unfoldingmentioning

confidence: 56%

Protein Folding Intermediates: Native-State Hydrogen Exchange

Bai

Sosnick

Mayne

et al. 1995

Science

1,062

1,396

View full text Add to dashboard Cite

The hydrogen exchange behavior of native cytochrome c in low concentrations of de-naturant 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.Under native conditions, a small fraction of any population of protein molecules occupies each possible higher energy, partially unfolded state, including even the fully unfolded state, as described by the Boltzmann distribution. The study of these partially unfolded forms (intermediates) may illuminate the fundamental cooperative nature of protein structure and define the unfolding and refolding pathways of a protein even though the intermediates are normally invisible to measurement. The energy levels and therefore the occupation of these conformationally excited states can be manipulated by denaturants and temperature. Hydrogen exchange experiments can then determine the hydrogens exposed in each higher energy form, their rates of exchange with solvent, and their sensitivity to the perturbant. From this we can infer, respectively, the structure, the free energy, and the surface exposure of each protein form.Results for cytochrome c reveal a small sequence of distinct partially unfolded forms with progressively increasing free energy and degree of unfolding. These appear to represent the major intermediates in the unfolding and refolding pathways of cytochrome c. Hydrogen exchange theoryExchangeable amide hydrogens (NH) that are involved in hydrogen-bonded structure can exchange with solvent hydrogens only when they are transiently exposed to solvent in some kind of closed to open reaction (1-3), as indicated in Eq. 1.(1)In the almost universally observed limiting case, referred to as EX2 (for bimolecular exchange) (1), the structural opening reaction enters the rate expression as a pre-equilibrium step. The exchange rate of any hydrogen, k ex , is then determined by its chemical exchange rate in the open form, k ch , multiplied by the equilibrium opening constant, K op (Eq. 2).

show abstract

Section: Global Local and Subglobal Unfoldingmentioning

confidence: 56%

Protein Folding Intermediates: Native-State Hydrogen Exchange

Bai

Sosnick

Mayne

et al. 1995

Science

1,062

1,396

View full text Add to dashboard Cite

show abstract

“…This approach can be extended to the unfolding reactions that determine protein hydrogen exchange (Kim & Woodward, 1993;Mayo & Baldwin, 1993;Bai et al, 1994a;Bai et al, 1994b;Qian et al, 1994). The data shown in Figure 2(right) for equine cytochrome c (cyt c ) include results from classical melting experiments at high GdmC1.…”

Section: The Physical Bases Of Protein Hydrogen Exchangementioning

confidence: 99%

Hydrogen exchange: The modern legacy of Linderstrøm‐Lang

et al. 1997

View full text Add to dashboard Cite

This discussion, prepared for the Protein Society's symposium honoring the 100th anniversary of Kaj Linderstr~m-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 modem structural biology.Keywords: cooperativity; folding intermediates; hydrogen exchange; Linderstrom-Lang; molten globule; protein foldingThe hydrogen exchange approach was conceived by Kaj Linderstram-Lang and implemented by him and his collaborators at the Carlsberg laboratories in the early 1950s. Pauling had just discovered the a-helix and P-sheet and postulated that they were stabilized by hydrogen bonds. In those exciting days at the dawn of modem protein science, Lang realized that peptide group NH hydrogens participate in continual exchange with the hydrogens of solvent, just as in the already understood polar side chains. He set out to test Pauling's ideas by measuring the exchange behavior of these hydrogens. Lang created entirely novel methods to measure H-D exchange, and together with his colleagues at the Carlsberg Labs, he studied hydrogen exchange in a number of proteins and peptides under various solution conditions (Hvidt & LinderstrgmLang, 1954, 1955a, 1955b Krause & Linderstrom-Lang, 1955;Linderstrom-Lang, 1955a, 1955b Berger & Linderstram-Lang, 1957; Benson & Linderstmm-Lang, 1959; Hvidt et ai., 1960). In comparison with modem capabilities the information available to Lang was severely limited in resolution and even in accuracy. Remarkably, he saw past these limitations and quickly moved past

show abstract

“…Two limits, or two mechanisms of exchange, have been proposed for the HX behavior of proteins: the EX2 limit, which generally describes the exchange in the native state or under relatively mild denaturing conditions (8)(9)(10), and the EX1 limit, which takes place at high temperature, high pH (g 7), or high concentration of denaturants (2). In the former case, a preequilibrium between open (exchangesusceptible) and closed (protected) conformations of amide protons exists; the observed exchange rates, k obs , reflect the rate constant k x for the base-catalyzed chemical exchange step H T D, subject to the thermodynamic equilibrium between open and closed states.…”

mentioning

confidence: 99%

“…In the former case, a preequilibrium between open (exchangesusceptible) and closed (protected) conformations of amide protons exists; the observed exchange rates, k obs , reflect the rate constant k x for the base-catalyzed chemical exchange step H T D, subject to the thermodynamic equilibrium between open and closed states. The pre-equilibrium is achieved by rapid changes between exchange-incompetent and exchange-competent conformations prior to the chemical exchange step (9). Such rapid motions have been attributed to both local fluctuations and global unfolding (11,12).…”

mentioning

confidence: 99%

“…In the EX1 case, on the other hand, the exchange is fast compared to the conformational motions that expose amide protons to solvent; and the slow process of conformational opening, or global unfolding, controls the observed rate. Adopting a kinetic scheme of the form (8)(9)(10)(11)5) where k op and k cl are the rate constants for the opening, and closing of protein conformations, the observed exchange rate k obs ) k op k x /(k cl + k x ) for k op << k cl reduces to k obs ) k op in the EX1 limit, given that k cl << k x . In the EX2 limit, on the other hand, k x << k cl , which leads to k obs ) K op k x .…”

mentioning

confidence: 99%

See 1 more Smart Citation

Correlation between Native-State Hydrogen Exchange and Cooperative Residue Fluctuations from a Simple Model

et al. 1998

View full text Add to dashboard Cite

Recently, we developed a simple analytical model based on local residue packing densities and the distribution of tertiary contacts for describing the conformational fluctuations of proteins in their folded state. This so-called Gaussian network model (GNM) is applied here to the interpretation of experimental hydrogen exchange (HX) behavior of proteins in their native state or under weakly denaturing conditions. Calculations are performed for five proteins: bovine pancreatic trypsin inhibitor, cytochrome c, plastocyanin, staphylococcal nuclease, and ribonuclease H. The results are significant in two respects. First, a good agreement is reached between calculated fluctuations and experimental measurements of HX despite the simplicity of the model and within computational times 2 or 3 orders of magnitude faster than earlier, more complex simulations. Second, the success of a theory, based on the coupled conformational fluctuations of residues near the native state, to satisfactorily describe the native-state HX behavior indicates the significant contribution of local, but cooperative, fluctuations to protein conformational dynamics. The correlation between the HX data and the unfolding kinetics of individual residues further suggests that local conformational susceptibilities as revealed by the GNM approach may have implications relevant to the global dynamics of proteins.Hydrogen exchange (HX) data for proteins directly indicate the relative accessibility of various protons to solvent, typically under conditions that can be denaturing to different extents. Observed HX times can vary over extremely broad ranges, from instantaneous to months. Generally these various times have been interpreted and ascribed to several kinds of processesslocal fluctuations for the fastest, local unfolding for intermediate, and global unfolding for the slowest exchanges (1). Some disagreements have arisen between the descriptions of the underlying kinetic processes and the interpretation of HX mechanisms because of differences in the operational definitions of these processes (2). HX from native proteins was recently pointed out to monitor with a remarkable precision rapid conformational fluctuations of the order of microseconds to milliseconds, while no correlation could be observed between HX free energies of individual residues and their unfolding rates which are several orders of magnitude slower than local fluctuations (3). Global unfolding rates are expected to be the same for all residues, while the superimposed exchange rates due to residue fluctuations vary depending on local interactions (4, 1, 5). Here we will avoid ascribing rates to specific processes but instead investigate directly the extent of protection in the native state and compare this with the measured exchange rates. This analysis has become possible with the development of a new simple model of protein structure and dynamics, recently proposed for analyzing the conformational fluctuations of folded proteins (6, 7).Two limits, or two mechanisms of ex...

show abstract

Protein Hydrogen Exchange in Denaturant: Quantitative Analysis by a Two-Process Model.

Cited by 58 publications

References 17 publications

Protein Folding Intermediates: Native-State Hydrogen Exchange

Protein Folding Intermediates: Native-State Hydrogen Exchange

Hydrogen exchange: The modern legacy of Linderstrøm‐Lang

Correlation between Native-State Hydrogen Exchange and Cooperative Residue Fluctuations from a Simple Model

Contact Info

Product

Resources

About