Microsecond protein folding kinetics from native-state hydrogen exchange.

Abstract

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.