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Hydrogen exchange and ligand binding: Ligand-dependent and ligand-independent protection in the Src SH3 domain

Department of Molecular and Cell Biology and QB3 Institute, University of California, Berkeley, California 94720-3206, USA

(RECEIVED July 14, 2004; FINAL REVISION August 30, 2004; ACCEPTED August 30, 2004)

Amide hydrogen-deuterium exchange has proven to be a powerful tool for detecting and characterizing high-energy conformations in protein ensembles. Since interactions with ligands can modulate these high-energy conformations, hydrogen exchange appears to be an ideal experimental probe of the physical mechanisms underlying processes like allosteric regulation. The chemical mechanism of hydrogen exchange, however, can complicate such studies. Here, we examine hydrogen exchange rates in a simple model system, the c-Src SH3 domain interacting with a short peptide ligand. Addition of ligand slows the rates of hydrogen exchange at nearly every amide for which we can obtain data. Careful analysis, however, reveals that this slowing is due primarily to a reduction in the population of free protein in the system, and not to any specific property of the complex. We present a method to separate the contributions of free and bound protein to the exchange kinetics that has allowed us to identify the subset of amides where exchange arises directly from the complex. These results demonstrate that the slowing of hydrogen exchange induced by ligand interactions should be interpreted with caution, and more extensive experiments are required to correlate changes in hydrogen exchange with changes in structure or internal dynamics.

Keywords: SH3; hydrogen exchange; ligand binding; protein dynamics

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04990205.

Reprint requests to: Susan Marqusee, 236 Hildebrand Hall, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3206, USA; e-mail: marqusee{at}berkeley.edu; fax: (510) 643-9290.

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