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Analysis of unfolding and protein dynamics in the regulatory domains of hematopoietic cell kinase with hydrogen exchange and mass spectrometry

John R Engen, University of Nebraska - Lincoln


The role of protein unfolding and protein dynamics in the conformation and activity of hematopoietic cell kinase (Hck), a signal transduction protein and oncogene, was investigated. The regulatory SH3 and SH2 domains were probed with hydrogen exchange (HX) and mass spectrometry (MS) to determine if changes in protein conformation and/or dynamics could play a role in enzymatic regulation. ^ Hck was analyzed in a stepwise fashion. This approach allowed comparison of how the domains related and how they influenced the conformation and dynamics of one another. Hydrogen exchange results for SH3 revealed that SH3 unfolded at pH 7, 25°C every 20 minutes. Unfolding was slowed by ligand binding. MS/MS techniques narrowed the region of unfolding. Slow unfolding in SH3 may be critical for Hck activation because the region of SH3 involved in unfolding is part of a potential conserved “north face” binding site for Hck regulators. ^ Binding SH2 to a high affinity ligand influenced HX into the folded form of SH2 and stiffened the structural dynamics throughout the domain. The later result may indicate that SH2 binding could be relayed to other parts of the protein by means of changes in protein flexibility. To test this hypothesis, the combination SH32 domain was incubated with either an SH3 or SH2 ligand and HX was monitored. The results indicated that communication between SH3 and SH2 was not facilitated by changes in SH3/SH2 motions. Interaction(s) of SH3/SH2 with the kinase domain may be more important as a regulatory mechanism rather than communication between SH3 and SH2 via dynamics changes. ^ The dynamics of the joint SH32 construct were compared to HX into the SH3 and SH2 domains when expressed alone. There proved to be significant changes in the dynamics of the domains when they are located next to each other. These results indicate that previous biophysical studies of isolated SH3 and SH2 domains may be misleading. The SH3 domain still unfolded in SH32 and at the same rate, further evidence that SH3 unfolding is a part of the dynamics of the whole protein and that may be critical for proper protein regulation. ^

Subject Area

Biology, Molecular|Chemistry, Biochemistry|Biophysics, General

Recommended Citation

Engen, John R, "Analysis of unfolding and protein dynamics in the regulatory domains of hematopoietic cell kinase with hydrogen exchange and mass spectrometry" (1999). ETD collection for University of Nebraska - Lincoln. AAI9952677.