Kinetics of unfolding and folding from amide hydrogen exchange in native ubiquitin.

Amide hydrogen (NH) exchange is one of the few experimental techniques with the potential for determining the thermodynamics and kinetics of conformational motions at nearly every residue in native proteins. Quantitative interpretation of NH exchange in terms of molecular motions relies on a simple two-state kinetic model: at any given slowly exchanging NH, a closed or exchange-incompetent conformation is in equilibrium with an open or exchange-competent conformation. Previous studies have demonstrated the accuracy of this model in measuring conformational equilibria by comparing exchange data with the thermodynamics of protein unfolding. We report here a test of the accuracy of the model in determining the kinetics of conformational changes in native proteins. The kinetics of folding and unfolding for ubiquitin have been measured by conventional methods and compared with those derived from a comprehensive analysis of the pH dependence of exchange in native ubiquitin. Rate constants for folding and unfolding from these two very different types of experiments show good agreement. The simple model for NH exchange thus appears to be a robust framework for obtaining quantitative information about molecular motions in native proteins.

Correlated motions in native proteins from MS analysis of NH exchange: evidence for a manifold of unfolding reactions in ovomucoid third domain.

Native-state amide hydrogen exchange monitored by NMR spectroscopy and mass spectrometry (MS) has the potential to provide detailed residue-level information regarding correlated motions occurring on the microseconds to seconds timescale. To expand the applicability of MS to these studies, a new algorithm has been developed to interpret MS data for exchange occurring between the EX2 and EX1 kinetic limits. Re-interpretation of MS data for ovomucoid third domain reveals multiple unfolding or partial unfolding reactions.

Microsecond to minute dynamics revealed by EX1-type hydrogen exchange at nearly every backbone hydrogen bond in a native protein.

A previous comprehensive analysis of the pH dependence of native-state amide hydrogen (NH) exchange in turkey ovomucoid third domain (OMTKY3) yielded apparent opening and closing rate constants (k(op) and k(cl)) at 14 NH groups involved in global conformational changes. This analysis has been extended to 18 additional slowly exchanging NH groups. Quench-flow experiments were performed to monitor NH exchange in native OMTKY3 from neutral to very alkaline pH ( approximately 12) conditions. Above pH 10 the mechanism of exchange switched from one governed by a rapid equilibrium preceding the chemistry of exchange (i.e. EX2 exchange), to one where exchange was limited by the rate of opening (i.e. EX1 exchange). Kinetics of solvent exposure are now known for nearly all backbone NH groups in native OMTKY3, yielding rate constants that span five orders of magnitude, 0.004 to 200 s(-1).

Defining protein ensembles with native-state NH exchange: kinetics of interconversion and cooperative units from combined NMR and MS analysis.

Previous studies of native-state peptide hydrogen atom (NH) exchange in turkey ovomucoid third domain (OMTKY3) yielded the thermodynamics and kinetics of unfolding and folding for the 14 slowest-exchanging peptide hydrogen atoms (NHs). Unfolding rate constants and free energies for nine of the NHs are very similar, suggesting that these NHs exchange during a single cooperative unfolding event. Electrospray ionization mass spectrometry (ESI-MS) has been used to test this hypothesis. ESI-MS data and MS peak simulations suggest that this hypothesis is incorrect: in spite of the similarity in their unfolding rate constants, only three to five of the nine residues exchange in a cooperative manner. Thus, residues with similar thermodynamics and kinetics of exchange are probably involved in multiple conformational equilibria. Overall, combined NMR and MS analysis of NH exchange provides a rich and complex picture of the ensemble properties of native proteins.

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.