Hydrogen Exchange Kinetics of Proteins in Denaturants: A Generalized Two-Process Model{ Hong Qian 1 * and Sunney I. Chan 2 1 Department of Applied Mathematics, University of Washington, Seattle WA 98195 USA 2 Noyes Laboratory of Chemical Physics, 127-72, California Institute of Technology Pasadena CA 91125, USA The recent progress in measurements on the amide hydrogen exchange (HX) in proteins under varying denaturing conditions, both at equili- brium and in transient relaxation, necessitates the development of a unifying theory which quantitatively relates the HX rates to the conformational energetics of the proteins. We present here a comprehen- sive kinetic model for the site-speci®c HX of proteins under varying solvent denaturing conditions based on the two-state protein folding model. The generalized two-process model considers both conformational ¯uctuations and residual protections, respectively, within the folded and unfolded states of a protein, as well as a global kinetic folding-unfolding transition between the two states. The global transition can be either rapid or slow, depending on the solvent condition for the protein. This novel model is applicable to the traditional equilibrium HX measure- ments in both EX2 and EX1 regimes, and also the recently introduced transient pulse-labeling HX experiments. A set of simple analytical equations is provided for quantitative interpretation of experimental data. The model emphasizes the use of full time-course of bi-exponential HX kinetics, rather than ®tting time-course data to single rate constants, to obtain quantitative information about ¯uctuating conformers within the folded and unfolded states of proteins. This HX kinetic model naturally unfolds into a simple two-state and two-stage kinetic interpret- ation for protein folding. It suggests that the various observed intermedi- ates of a protein can be interpreted as dominant isomers of either the folded or the unfolded state under different solvent conditions. This simple, minimalist's view of protein folding is consistent with various recent experimental observations on folding kinetics by HX. # 1999 Academic Press Keywords: protein folding; intermediates; local ¯uctuations; molten- globule; pulse-labeling *Corresponding author Introduction Due to the highly cooperative nature of protein structures, the simplest model for protein folding in aqueous solution considers only two states: a folded state in which a polypeptide ¯uctuates among an ensemble of highly ordered structures, and an unfolded state in which the polypeptide adopts a large number of random conformations. This two-state model hypothesizes that the confor- mational transitions within each state are fast, but the transitions between the two states are relatively slow (Creighton, 1988; Zwanzig, 1997). In the language of energy landscape, the latter is equival- ent to asserting that conformers within each state are separated by relatively low energy barriers, while the one separating the two sets of states is substantial in height. In other words, the two mol- ecular populations are well separated both structu- rally and energetically. Contrary to the crystal- lographic view of proteins, however, the highly ordered structures still have signi®cant confor- mational ¯exibility. Under or near the ``native'' condition, a protein ¯uctuates among a wide spec- trum of conformations, ranging from small local ¯uctuations to major conformational changes, even all the way to the globally unfolded forms. The {Dedicated to Dr Andrew Morton, who tragically passed away on July 17, 1998, for the living memory of many long discussions on hydrogen exchange kinetics and protein folding intermediates. Abbreviations used: HX, hydrogen exchange; CD, circular dichroism. Email address of the corresponding author: qian@amath.washington.edu Article No. jmbi.1998.2484 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 286, 607±616 0022-2836/99/070607±10 $30.00/0 # 1999 Academic Press