Hydrogen exchange kinetics of proteins in denaturants: A generalized two-process model

The recent progress in measurements on the amide hydrogen exchange (HX) in proteins under varying denaturing conditions, both at equilibrium and in tr ansient relaxation, necessitates the development of a unifying theory which quantitatively relates the HX rates to the conformational energetics of th e proteins. We present here a comprehensive kinetic model for the site-spec ific HX of proteins under varying solvent denaturing conditions based on th e two-state protein folding model. The generalized two-process model consid ers both conformational fluctuations and residual protections, respectively , within the folded and unfolded states of a protein, as well as a global k inetic folding-unfolding transition between the two states. The global tran sition can be either rapid or slow, depending on the solvent condition for the protein. This novel model is applicable to the traditional equilibrium HX measurements in both EX2 and EX1 regimes, and also the recently introduc ed transient pulse-labeling HX experiments. A set of simple analytical equa tions is provided for quantitative interpretation of experimental data. The model emphasizes the use of full time-course of bi-exponential HX kinetics , rather than fitting time-course data to single rate constants, to obtain quantitative information about fluctuating conformers within the folded and unfolded states of proteins. This HX kinetic model naturally unfolds into a simple two-state and two-stage kinetic interpretation for protein folding . It suggests that the various observed intermediates of a protein can be i nterpreted as dominant isomers of either the folded or the unfolded state u nder different solvent conditions. This simple, minimalist's view of protei n folding is consistent with various recent experimental observations on fo lding kinetics by HX. (C) 1999 Academic Press.