Hydrogen exchange shows peptide binding stabilizes motions in Hck SH2

Src-homology-2 domains are small, 100 amino acid protein modules that are p resent in a number of signal transduction proteins. Previous NMR studies of SH2 domain dynamics indicate that peptide binding decreases protein motion s in the pico- to nanosecond, and perhaps slower, time-range. We suggest th at amide hydrogen exchange and mass spectrometry may be useful for detectin g changes in protein dynamics because hydrogen exchange rates are relativel y insensitive to the time domains of the dynamics. In the present study, hy drogen exchange and mass spectrometry were used to probe hematopoietic cell kinase SH2 that was either free or bound to a 12-residue high-affinity pep tide. Hydrogen exchange rates were determined by exposing free and bound SH 2 to D2O, fragmenting the SH2 with pepsin, and determining the deuterium le vel in the peptic fragments. Binding generally decreased hydrogen exchange along much of the SH2 backbone, indicating a widespread reduction in dynami cs. Alterations in the exchange of the most rapidly exchanging amide hydrog ens, which was detected following acid quench and analysis by mass spectrom etry, were used to locate differences in low-amplitude motion when SH2 was bound to the peptide. In addition, the results indicate that hydrogen excha nge from the folded form of SH2 is an important process along the entire SH 2 backbone.