RUI: Folding Kinetic Studies of Permuted Myoglobins

Western Washington University, Bellingham WA

Investigators

Abstract

An understanding of the principles that determine protein folding and stability is critical for guiding the rational design of proteins with novel functional properties, and for accurate predictions of tertiary structure from amino acid sequence. This project will increase our understanding of the relationship between altered topology and the folding pathway for a protein, and provide experimental data that can be used to refine structure prediction algorithms. The specific aims are: (1) to compare the folding and unfolding rates of wild-type and three to four circularly permuted sperm whale myoglobins using stopped-flow circular dichroism spectroscopy; (2) to map differences in the transition state structures for wild-type and circularly permuted myoglobins using perturbation methods. These studies entail generation and bacterial expression of genes encoding permuted myoglobin proteins (permuteins) as well as site-directed mutants of the permuteins and the wild-type myoglobin. Purified apoproteins will be analyzed for stability to chemical denaturation and in kinetic assays for folding and unfolding. CD spectroscopy will be used to derive the stability parameters and kinetic studies will be carried out with stopped-flow CD to measure rates of permutein folding and unfolding at various denaturant concentrations. Kinetic and equilibrium stability data will be used to map the relative energies of the denatured, native and transition states for folding reactions. The long-term goals of this research include a thorough determination of those regions of the myoglobin sequence that will tolerate topological mutation, and characterization of the folding pathway for these mutants. This project will provide an opportunity for several undergraduate students to integrate classroom teaching in biochemistry and biophysics with an intensive research experience. The students will carry out all aspects of the proposed work from cloning genes to CD studies, as well as presentation of results at local and national meetings. Hands-on experience with equilibrium and kinetic data collection and interpretation will greatly enhance student comprehension of biophysical principles, and prepare these students well for further study in biochemistry.