Capturing protein dynamics by time-resolved spectroscopy: Folding and electron tunneling in cytochromes

Abstract

We have resolved the folding kinetics of two c-type cytochromes, one that exhibits twostate folding and one that has an on-pathway folding intermediate. We resolve millisecond-timescale folding by coupling time-resolved fluorescence energy transfer (FRET) with a continuous flow mixer. The efficiency of energy transfer between a dansyl label, attached to single-cysteine mutants, and the cytochrome heme during the folding process provides us with time-dependent distance distributions, which provide information about the kinetics and mechanism of folding.We are also interested in characterizing the pathway dependence of electron tunneling rates between metal sites in proteins. We have converted a b-type cytochrome to a c-type cytochrome by covalently linking the porphyrin to cysteine residues. We investigate the effects of these changes to the protein structure, i.e., increased rigidity and potential new equatorial tunneling pathways, on the electron transfer rates, measured via transient absorption, in a series of Ru-modified proteins.