Protein folding, stability, and assembly: A thorough assessment of complex behavior
Wilson, Corey J.
Matthews, Kathleen S.
Doctor of Philosophy
Proteins play an integral role in virtually every biological process. The function of a protein is determined by its three-dimensional structure. However, the connection between a linear sequence of amino acids and its biologically viable conformation is not well understood. Understanding protein folding, stability, and assembly is important because (i) many diseases are associated with the failure of proteins to fold correctly or to remain folded under physiological conditions and (ii) this understanding is requisite for the de novo design of new proteins with prescribed properties constructed from first principles using their coding sequences. To shed light on the fold/stability/assembly relationship, two model proteins were investigated---the metalloprotein azurin and lactose repressor---that display vastly different properties. Experimental and theoretical/computational approaches were combined to assess the protein-folding dynamics of a large, multidomain protein with complex topology. A minimalist protein model reproduces remarkably well the complex folding process observed for a monomeric variant of the lactose repressor protein. Moreover, theoretical results were compared to the experimental derived folding dynamics by using the structures to estimates experimental parameters. A series of studies employing both experiment and computation were conducted to assess the role of cofactors in protein folding and stability. The role of specific structural determinants in the folding of Pseudomonas aeruginosa azurin were assessed as well as the effects of a specific cofactor on the transition state ensemble and mechanism of the folding process. Taken together, this body of work illustrates the synergy in combining robust theoretical/computational frameworks with objective experimentation and sets the stage for the development of consolidated approaches that are of direct biomedical relevance.
Molecular biology; Biochemistry; Biophysics