IDENTIFICATION AND LOCALIZATION OF SLOW, NATURAL, COOPERATIVE UNFOLDING IN THE HEMATOPOIETIC-CELL KINASE SH3 DOMAIN BY AMIDE HYDROGEN-EXCHANGE AND MASS-SPECTROMETRY

Protein unfolding on a fast time scale (milliseconds-minutes) has been widely reported, but slower unfolding events (10 min-hours) have rece ived less attention. amide hydrogen exchange (HX) and mass spectrometr y (MS) were used to investigate the unfolding dynamics of the hematopo ietic cell kinase (Hck) SH3 domain. analysis of mass spectra after deu terium exchange into intact Hck SH3 indicates a cooperative unfolding event involving 24-61% of the domain and occurring with a half-life of approximately 20 min under physiological conditions. To identify the unfolding region, SH3 was incubated in D2O and proteolytically fragmen ted into peptides that were analyzed by mass spectrometry. Correlation of HX rates and isotope patterns reveals cooperative unfolding in sev eral regions, including the C-terminal half of the RT-loop and a beta- sheet flanking the binding site, Binding of a prolyl-rich segment from the HIV Nef protein slows unfolding by a factor of 3. Further analysi s yields a K-D of 25 mu M for the Nef peptide. These results demonstra te that an inherent flexibility in the SH3 domain may assist interconv ersion of the closed, intramolecularly ligated state and the open, act ive state of Src family kinases. Furthermore, this type of previously undetectable, slow unfolding process may provide the basis for new mec hanisms in which kinetics of local unfolding combines with thermodynam ics to regulate enzymatic activity. The combination of hydrogen exchan ge and mass spectrometry appears to be the only general method capable of examining these slow unfolding processes.