March 5, 2010
INSIGHTS INTO MEMBRANE PROTEIN STABILITY AND FOLDING KINETICS VIA NOVEL SINGLE-TRYPTOPHAN MUTANTS
MARC U*STAR Fellow
Summer Research Site: UC San Diego
Abstract Membrane proteins adopt unique three-dimensional structures in order to be biologically active. Protein misfolding can lead to a variety of human diseases such as AlzheimerÂs, ParkinsonÂs, Creutzfeldt-JakobÂs and type II diabetes. This research project focuses on the thermodynamics and kinetics of membrane protein folding using the integral membrane protein Outer Membrane Protein A (OmpA) as a model system. We have generated a novel mutant W15 to study the OmpA folding. The spectroscopic properties of Trp reflect the local environment, and this sensitivity can be utilized to monitor protein folding. The W15 and other mutants of OmpA were prepared by site-directed mutagenesis, isolated, and purified by chromatography. Protein concentrations and purity were determined by UV-VIS spectroscopy. Steady state tryptophan fluorescence spectroscopy was used to monitor the equilibrium between folded and unfolded states of OmpA mutants. These results help establish a foundation for in depth studies on membrane protein folding.