Event Information

Pooja Arora (Duke University)
Biophysical Characterization of Folding of IgG Binding Domains of Protein A
Abstract:
The interconversion between the native (N) and denatured (D) states of a protein is coupled to its various biological activities, such as enzymatic functions, translocation, and degradation. Understanding the underlying energetics and kinetics of the folding and unfolding pathways is essential for dissecting both protein folding and function in the cell. In this thesis, we characterize the folding biophysics of a small protein, the B-domain of protein A (BdpA). We show that the fast folding of BdpA (kf 120,000sec-1 at 37˚C) is not evolutionarily maximized, and can be accelerated by a single amino acid substitution designed specifically to stabilize a secondary structural element of BdpA. Based on the fastest folding rate constant of BdpA (kf 249,000sec-1 at 45˚C), using laser-induced temperature jump experiments, we suggest a new lower-limit for the pre-exponential factor for the protein folding reaction.

Equilibrium results using CD suggest that BdpA exhibits significant residual electrostatic interactions in D. The weak pH- and ionic strength-dependence of the equilibrium stability indicates that ionizable residues in BdpA are involved in similar local, favorable Coulombic interactions in N and D. These findings are interesting since the theoretical and experimental models often assume the presence of interactions or structure to be negligible in D.

To understand the folding behavior of BdpA in the five-domain protein A, we characterized the folding of a multi-domain protein containing two BdpA sequences in tandem (BBdpA). Based on the experimentally insignificant thermodynamic and kinetic coupling in BBdpA, we suggest a plausible mechanism for the efficient secretion of full-length protein A. We also derive theoretical models that will provide experimentalists with a framework to analyze and interpret the equilibrium and kinetic data of proteins with multiple, uncoupled domains.

Ph.D. Defense Examination Seminar

Wednesday, April 19, 2006, 2:15pm
Student Exams Seminar