title: Hydrogen exchange and ligand binding: ligand-dependent and ligand-independent protection in the Src SH3 domain.
abstract: 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.
authors.affiliation: QB3 Institute, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3206, USA.
authors.forename: David
authors.initials: D
authors.lastname: Wildes
authors.forename: Susan
authors.initials: S
authors.lastname: Marqusee
mesh_terms: D000818:Animals; D001665:Binding Sites; D002645:Chickens; D006859:Hydrogen / Q000737:chemistry*; D008024:Ligands; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000737:chemistry; D018909:src Homology Domains
chemical_list: D008024:Ligands; D010455:Peptides; D006859:Hydrogen; D016392:Proto-Oncogene Proteins pp60(c-src)