Protein-protein interactions and dynamics probed by hydrogen/deuterium exchange

Work presented within focuses on applications of amide H/D exchange experiments to map sites of conformational and dynamic changes in proteins upon substrate binding or modification. Amide exchange coupled with mass spectrometry provides a powerful tool to view the solvent accessibility of regions of a protein in its native state. The solvent accessibility of amides vary greatly depending on whether the amide is participating in stable hydrogen bonds in the interior core of a globular protein, or is part of an unstructured surface peptide. Studies presented here show that amides exchanging on the seconds to minute time-scale serve as useful probes for localizing allosteric changes upon protein-ligand complexation, or post-translational modification.; Chapter II demonstrates the sensitivity of the technique by mapping changes in the interdomain interface of the chemotaxis protein methylesterase CheB. Information about the activated state of CheB has been hindered because of the transient nature of the Asp 56 phosphorylation event (t_1/2 = 1 sec). Comparison of results from phosphorylated and unphosphorylated full length CheB, and from the catalytic domain fragment of CheB (residues 147–349) reveal that most of the interdomain surface is maintained and only subtle rearrangements are needed to confer a catalytically enhanced protein.; In Chapter III, solvent accessibility changes which occur upon active site occupation of the blood coagulation protein, thrombin, are mapped. Development of a quenched-flow protocol, which enables capture of early time points in H/D exchange, aided our ability to observe allosteric sites of change for key binding surface loops. Results indicate a path of communication exists between the active site and remote co-factor binding regions.; In Chapter IV, the quenched-flow system was used to measure H/D exchange kinetics for the non-globular protein, IκBα. Initial characterization of the protein indicates that only part of IκBα is folded, and that the remainder of the molecule folds upon binding to NFκB. However, future studies are proposed to study the solvent accessibility changes of IκBα when studied in complex with NFκB.; In Chapter V, future experiments will be presented including method development ideas to improve the resolution of the amide exchange experiments for mass spectrometry.