Native state dynamics drive the unfolding of the SH3 domain of PI3 kinase at high denaturant concentration.
Little is known about the role of protein dynamics in directing protein unfolding along a specific pathway and about the role played by chemical denaturants in modulating the dynamics and the initiation of unfolding. In this study, deuterium-hydrogen exchange (HX) detected by electrospray ionization mass spectrometry (ESI-MS) was used to study the unfolding of the SH3 domain of the PI3 kinase. Unfolding on the principal unfolding pathway occurs in 2 steps, both in the absence and in the presence of 1.8 M guanidine hydrochloride (GdnHCl). In both cases, the first step leads to the formation of an intermediate, I(N), with 5 fewer protected amide hydrogen sites than in N. In the second step, I(N) loses the structure protecting the remaining 14 amide hydrogen sites from HX as it unfolds completely. ESI-MS analysis of fragments of the protein created by proteolytic digestion, after completion of the HX reaction, shows that I(N) has lost protection against HX in the same segments of native structure during unfolding in the absence and presence of 1.8 M GdnHCl. Hence, GdnHCl does not appear to play a direct active role in the initiation of unfolding. However, at higher GdnHCl concentrations, a second unfolding pathway is shown to compete effectively with the N <--> I(N) <--> U pathway. In this way, the denaturant modulates the energy landscape of unfolding.
Wani
Ajazul Hamid
AH
National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India.
Udgaonkar
Jayant B
JB
D003903:Deuterium / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D004795:Enzyme Stability / Q000187:drug effects; D019791:Guanidine / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007700:Kinetics; D013058:Mass Spectrometry; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation / Q000187:drug effects*; D017510:Protein Folding / Q000187:drug effects*; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D018909:src Homology Domains
D010455:Peptides; D003903:Deuterium; D019869:Phosphatidylinositol 3-Kinases; D019791:Guanidine