Journal of Molecular Biology
GuHCl and NaCl-dependent Hydrogen Exchange in MerP Reveals a Well-defined Core with an Unusual Exchange Pattern
Introduction
One of the principal goals of protein folding studies is to establish how amino acid sequences encode the three-dimensional structures of proteins. Analysis of the kinetics of hydrogen exchange in proteins is a powerful method for studying both the structural and dynamic properties of the protein structure.1, 2, 3, 4, 5, 6 Hydrogen–deuterium exchange experiments are useful to characterise partially unfolded states7, 8, 9, 10 that exist at energy levels between the native and denatured states,11, 12 thus providing valuable information about the properties of the protein-folding landscape.13
Equilibrium denaturation of oxidised MerP by guanidine hydrochloride (GuHCl) is fully reversible, following a two-state model, and its equilibrium unfolding parameters have been determined.14 The oxidised form of MerP was chosen for the present study because it can be obtained in a homogenous form. In the oxidised MerP, one disulphide bridge is formed between the two adjacent amino acid residues (Cys14 and Cys17) that exclude binding of Hg2+ because binding require the reduced form of the cysteine residues. In previous MerP refolding experiments, no evidence has been acquired for a burst or hidden phase that could account for any accumulation of an intermediate preceding the rate-limiting formation of the native state.14 However, native-state hydrogen exchange analysis can reveal hidden intermediates that may not be detected when using conventional kinetic methods.15 Such kinds of intermediates have been identified successfully for cytochrome c3 and Rd-apocyt b562.16
Despite the fact that MerP appears to fold in a two-state manner, a recent hydrogen exchange study of MerP at different temperatures in the pre-transitional region has revealed partly unfolded states of MerP that differ in stability.17 In the study reported here, the exchange behaviour of MerP has been studied as a function of the concentration of GuHCl in the range associated with the pre-unfolding transition region. We were able to determine the free energy values in the absence of denaturant for individual amide protons, as well as the free energy values for the GuHCl-dependent unfolding process. For one set of amide protons, there is a weak dependence on denaturant, indicating that the dominant mechanism for exchange under native conditions involves local fluctuations with the exposure of little new surface. Most of these residues have a low level of stability and are found in loops and at the edges of secondary structure elements. For another set of the amide protons, a linear denaturant-dependence is observed associated with m-values between 1.3 kcal mol−1 M−1 and 3.1 kcal mol−1 M−1. Thus, a large portion of the molecule must be involved in the unfolding event that allows exchange at these residues because it is accompanied by the exposure of a considerable amount of new surface area. Interestingly, for 16 of the amide protons, located in a well-defined core of the secondary structure, the free energy of exchange versus [GuHCl] plots have pronounced upward curvature. Notably, this feature of non-linearity is detected from analysis of H/2H exchange experiments exclusively, and not from comparisons between folding experiments performed with different methods. This finding may indicate that the central core of the protein, when unfolded in H2O, is up to five times more resistant to exchange, and thus better protected than the same core of the protein when unfolded at concentrations of GuHCl higher than 0.5 M. However, to investigate if the observed upward curvature was dependent on the guanidinium or chloride ions, hydrogen exchange experiments were conducted with different concentrations of NaCl. Notably, the chloride ions (Cl−) have no stabilizing effect on a large part of the protein structure but, interestingly, show a pronounced stabilizing effect on the cluster that was identified with upward curvature in the GuHCl experiments. A thorough analysis show that both guanidinium and chloride ions contribute to the observed curvature in the hydrogen exchange experiment.
Section snippets
Theory
The exchange rates were analysed according to the following scheme:18, 19
A general property of the transition NHclosed to NHopen is that it leads to solvent-exposure of the proton. Such exposure may result from several different types of event, for example, local structural fluctuations in the folded protein or cooperative unfolding of larger portions of the molecule.
Under the conditions used in our experiments, proton exchange occurs via an EX2
Results
MerP contains 72 amino acid residues, three of which are proline. Thus, 68 NH protons are visible in the 2D 1H NMR spectrum.27 The H/2H exchange behaviour of these protons was measured at different [GuHCl] to gain information on the dynamics and stability of structural elements.
Discussion
The results from both GuHCl and NaCl titrations are analysed here. The discussion begins with an analysis of the distribution in stabilities for the individual residues and their m-values obtained from GuHCl-dependent exchange. In the second section we discuss residues for which the exchange mechanism is dominated by local fluctuation. In the third section we make a detailed analysis about the upward curvature phenomenon, which was observed in the GuHCl titration for 16 residues in the protein.
Preparation and purification of the protein
Escherichia coli cells were grown in minimal medium (6 g/l of Na2HPO4, 3 g/l of KH2PO4, 0.5 g/l of NaCl, 10 ml/l of 40% (w/v) glucose, 0.01 mM FeSO4, 0.28 mM K2SO4, 0.5 μM CaCl2, 1 mM MgCl2, 1 ml/l of micronutrients, 1 μg/l of vitamin mixture, 0.5 g/l of 15NH4Cl) containing 100 μl/l of ampicillin. The 15N-labelled oxidised MerP was purified as described.52 The solution of oxidized protein was dialyzed against water/HCl at pH 5.0 and lyophilised.
H/2H exchange experiment
To investigate whether addition of GuHCl causes any shift
Acknowledgements
This work was supported by a grant from J C Kempes Minnes Stipendiefond to A.-C.B., and a grant from the Swedish Natural Science Research Council to B.-H.J. (K5104-5999). We thank Dr M. Oliveberg for valuable discussions and Ms Katarina Wallgren for excellent technical assistance.
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