Erin Cunningham

currently a postdoc at UC Berkeley

Examining the Mechanism and Specificity of α-Lytic Protease and S. griseus Protease B Pro Region Mediated Folding

My thesis work focuses on understanding the pro region mediated folding of α-lytic protease. Although the general characteristics of α-lytic protease (α-LP) folding are well established, the basis for the large folding barrier and the mechanism whereby the pro region circumvents this barrier remain unclear. I am using a variety of pro region variants and a homologous protease system (S. griseus protease B, or SGPB) to better understand these issues.

Specifically, I am:

1) characterizing truncated/mutated pro regions and their effect on α-LP folding and inhibition,

2) investigating the independent folding of the N-and C-terminal domains of α-LP in the absence and the presence of pro region and in collaboration with another graduate student, Stephanie Truhlar,

3) characterizing SGPB folding and examining pro region specificity in SGPB and α-LP folding.

Recent results from my work provide details that refine our proposed model of Pro-mediated folding of α-LP, as depicted in the figure. Characterization of several Pro mutations within the Pro-α-LP interface reveals that while the Pro N-domain is not involved in initial binding to the protease (step b), it is important in binding and stabilizing the folding transition state (step c). Furthermore, studies of Pro degradation by α-LP identify the secondary cleavage site within Pro (step e), define the fragmentation of Pro that leads to its proteolytic removal (step f) and show that α-LP is capable of degrading Pro without the assistance of other exogenous proteases.

Publications

"Kinetic stability as a mechanism for protease longevity," Cunningham, E.L., Jaswal, S.S., Sohl J.L., and Agard, D.A., Proc. Nat. Acad. Sci., 1999, 96, 11008-11014. pdf.