Brian Kelch

currently a postdoc at UC Berkeley

graduated 2007

It is commonly assumed that the native three-dimensional structure of a protein is the lowest free energy state and that all the information necessary to reach this native structure is entirely contained within its primary sequence. However, studies of alpha-lytic protease (aLP) have shown that these assumptions are not always valid. aLP requires an additional pro region, supplied either in trans or in cis, to traverse an extremely large energetic barrier to reach the native state. The native state itself is extremely stable kinetically (t1/2, unfolding ~ 1.2 years), but is actually less stable thermodynamically than the fully unfolded form by greater than 3kcal/mol. Despite high-resolution structures of both the mature protease and the protease in complex with the pro region and various dynamics studies, we still do not understand the structural basis for the large unfolding barrier in aLP. Since the cause of the extraordinarily high barrier to unfolding is still largely unknown, analysis of this extreme example may facilitate a deeper understanding of the source of the barrier and the nature of unfolding barriers in general. I propose to use mutagenesis and directed evolution strategies of aLP in conjunction with the analysis of a thermophilic homologue of aLP (T. fusca protease A) to gain insight into this issue.

Research Goals

1) Obtain mutants of alpha-lytic protease (aLP) that have either increased or decreased barriers to unfolding.

2) Characterize the folding energetics of interesting aLP mutants and of a thermophilic aLP homologue, TFPA. Obtain X-ray crystal structure of TFPA.

3) Characterize the dynamics of the aLP folding mutants using HX NMR.

4) Test hypotheses for the basis of kinetic stability by rational mutagenesis of aLP and/or other aLP homologues.

Publications

Brian A. Kelch, Kyle P. Eagen, F. Pinar Erciyas, Elisabeth L. Humphris, Adam R. Thomason, Shinji Mitsuiki and David A. Agard, "Structural and Mechanistic Exploration of Acid Resistance: Kinetic Stability Facilitates Evolution of Extremophilic Behavior," Journal of Molecular Biology, 2007 Mar (pdf).

Fuhrmann, C.N., Kelch, B.A., Ota, N., Agard, D.A. "The 0.83Å resolution crystal structure of -lytic protease reveals the detailed structure of the active site and identifies a source of conformational strain," J. Mol Biol. (2004);338(5):999-1013. (html or pdf)