Unfolded states of heme proteins

Abstract

Cytochromes adopt discrete structures when the heme cofactor is present. Since the heme provides crucial contacts in the native protein, this hydrophobic prosthetic group plays an important role in the folding mechanism and introduces interesting features into the folding landscape. Since the iron-porphyrin is sensitive to protein structure and environment, it also acts an optical probe and will be used in photophysical and photochemical studies of three alpha-helical proteins in the electron transport family of cytochromes: both horse heart and Saccharomyces cerevisiae iso-1 cytochrome c (cyt c), Rhodopseudomonas palustris cytochrome c’ (cyt c’), and the engineered cytochrome cb562 (cyt cb562) from E. coli cytochrome b562. The work in this thesis expands upon previous research in the Gray Group that has shown heterogenous unfolded populations in these three alpha-helical proteins. Triplet state decay kinetics of Zn-substituted cytochrome c (Zn-cyt c) reveal the level of solvent exposure of the heme through an isotope effect and bimolecular quenching of lifetimes. Differences of the GuHCl, urea, and thermally-induced unfolded states between Zn- and Fe-cyt c, cyt c’ and cb562 were uncovered, revealing a distinct hydrophobic effect. The inability of elevated temperature to disrupt hydrophobic pockets was studied further for DNS(C102)-cyt c through FET kinetics that reveal a significant compact population at high temperature. Finally, initial experiments on macromolecular crowding of cyt c revealed only a slight effect on the equilibrium unfolded states.