Hydrogen exchange shows peptide binding stabilizes motions in Hck SH2.
Src-homology-2 domains are small, 100 amino acid protein modules that are present in a number of signal transduction proteins. Previous NMR studies of SH2 domain dynamics indicate that peptide 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 changes in protein dynamics because hydrogen exchange rates are relatively insensitive to the time domains of the dynamics. In the present study, hydrogen exchange and mass spectrometry were used to probe hematopoietic cell kinase SH2 that was either free or bound to a 12-residue high-affinity peptide. Hydrogen exchange rates were determined by exposing free and bound SH2 to D(2)O, fragmenting the SH2 with pepsin, and determining the deuterium level in the peptic fragments. Binding generally decreased hydrogen exchange along much of the SH2 backbone, indicating a widespread reduction in dynamics. Alterations in the exchange of the most rapidly exchanging amide hydrogens, which was detected following acid quench and analysis by mass spectrometry, were used to locate differences in low-amplitude motion when SH2 was bound to the peptide. In addition, the results indicate that hydrogen exchange from the folded form of SH2 is an important process along the entire SH2 backbone.
Department of Chemistry, University of Nebraska-Lincoln 68588-0304, USA.
J R
JR
Engen
W H
WH
Gmeiner
T E
TE
Smithgall
D L
DL
Smith
D000595:Amino Acid Sequence; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism*; D051572:Proto-Oncogene Proteins c-hck; D011522:Protons; D013816:Thermodynamics; D018909:src Homology Domains
D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D011522:Protons; D011505:Protein-Tyrosine Kinases; C496631:HCK protein, human; D051572:Proto-Oncogene Proteins c-hck