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Mapping protein energy landscapes with amide hydrogen exchange and mass spectrometry: I. A generalized model for a two-state protein and comparison with experiment

Hui Xiao¹, Joshua K. Hoerner¹, Stephen J. Eyles², Andras Dobo¹, Edward Voigtman¹, Andre I. Mel’uk² and Igor A. Kaltashov¹

¹ Department of Chemistry and ² Department of Polymer Science & Engineering, University of Massachusetts at Amherst, Amherst, Massachusetts 01003, USA

(RECEIVED July 21, 2004; FINAL REVISION September 16, 2004; ACCEPTED October 9, 2004)

Protein amide hydrogen exchange (HDX) is a convoluted process, whose kinetics is determined by both dynamics of the protein and the intrinsic exchange rate of labile hydrogen atoms fully exposed to solvent. Both processes are influenced by a variety of intrinsic and extrinsic factors. A mathematical formalism initially developed to rationalize exchange kinetics of individual amide hydrogen atoms is now often used to interpret global exchange kinetics (e.g., as measured in HDX MS experiments). One particularly important advantage of HDX MS is direct visualization of various protein states by observing distinct protein ion populations with different levels of isotope labeling under conditions favoring correlated exchange (the so-called EX1 exchange mechanism). However, mildly denaturing conditions often lead to a situation where the overall HDX kinetics cannot be clearly classified as either EX1 or EX2. The goal of this work is to develop a framework for a generalized exchange model that takes into account multiple processes leading to amide hydrogen exchange, and does not require that the exchange proceed strictly via EX1 or EX2 kinetics. To achieve this goal, we use a probabilistic approach that assigns a transition probability and a residual protection to each equilibrium state of the protein. When applied to a small protein chymotrypsin inhibitor 2, the algorithm allows complex HDX patterns observed experimentally to be modeled with remarkably good fidelity. On the basis of the model we are now in a position to begin to extract quantitative dynamic information from convoluted exchange kinetics.

Keywords: protein dynamics; protein energy landscape; protein equilibrium states; amide hydrogen exchange; mass spectrometry

Abbreviations: CI2, chymotrypsin inhibitor 2 • ESI, electrospray ionization • EX1, exchange regime 1 • EX2, exchange regime 2 • EXX, intermediate exchange regime • HDX, hydrogen/deuterium exchange • MS, mass spectrometry • N, native state • U, unfolded state

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041001705.

Reprint requests to: Igor A. Kaltashov, Department of Chemistry, University of Massachusetts at Amherst, 710 North Pleasant Street, LGRT#701 Amherst, MA 01003, USA; e-mail: kaltashov{at}chem.umass.edu; fax: (413) 545-4490.

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