| Abstract
| - Src-homology-2 domains are small, 100 amino acid protein modules that are present in anumber of signal transduction proteins. Previous NMR studies of SH2 domain dynamics indicate thatpeptide binding decreases protein motions in the pico- to nanosecond, and perhaps slower, time range.We suggest that amide hydrogen exchange and mass spectrometry may be useful for detecting changesin protein dynamics because hydrogen exchange rates are relatively insensitive to the time domains of thedynamics. In the present study, hydrogen exchange and mass spectrometry were used to probe hematopoieticcell kinase SH2 that was either free or bound to a 12-residue high-affinity peptide. Hydrogen exchangerates were determined by exposing free and bound SH2 to D2O, fragmenting the SH2 with pepsin, anddetermining the deuterium level in the peptic fragments. Binding generally decreased hydrogen exchangealong much of the SH2 backbone, indicating a widespread reduction in dynamics. Alterations in theexchange of the most rapidly exchanging amide hydrogens, which was detected following acid quenchand analysis by mass spectrometry, were used to locate differences in low-amplitude motion when SH2was bound to the peptide. In addition, the results indicate that hydrogen exchange from the folded formof SH2 is an important process along the entire SH2 backbone.
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