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 received less attention. Amide hydrogen exchange (HX) and mass spectrometry (MS) were used to investigate the unfolding dynamics of the hematopoietic cell kinase (Hck) SH3 domain. Analysis of mass spectra after deuterium 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 fragmented into peptides that were analyzed by mass spectrometry. Correlation of HX rates and isotope patterns reveals cooperative unfolding in several 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 analysis yields a KD of 25 microM for the Nef peptide. These results demonstrate that an inherent flexibility in the SH3 domain may assist interconversion of the closed, intramolecularly ligated state and the open, active state of Src family kinases. Furthermore, this type of previously undetectable, slow unfolding process may provide the basis for new mechanisms in which kinetics of local unfolding combines with thermodynamics to regulate enzymatic activity. The combination of hydrogen exchange and mass spectrometry appears to be the only general method capable of examining these slow unfolding processes.
Engen
J R
JR
Department of Chemistry, University of Nebraska-Lincoln 68588-0304, USA.
Smithgall
T E
TE
Gmeiner
W H
WH
Smith
D L
DL
D000577:Amides; D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D003001:Cloning, Molecular; D004926:Escherichia coli; D015693:Gene Products, nef / Q000737:chemistry; D006678:HIV / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010434:Pepsin A; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism*; D051572:Proto-Oncogene Proteins c-hck; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D054311:nef Gene Products, Human Immunodeficiency Virus; D018909:src Homology Domains
D000577:Amides; D015693:Gene Products, nef; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D011505:Protein-Tyrosine Kinases; C496631:HCK protein, human; D051572:Proto-Oncogene Proteins c-hck; D010434:Pepsin A