Conformational Dynamics of a Fast Folding Cytochrome Captured by Time-Resolved Spectroscopy

Abstract

We probe intrachain contact dynamics in unfolded cytochrome cb562 by monitoring heme quenching of excited ruthenium photosensitizers covalently bound to residues along the polypeptide. Tertiary contact formation kinetics provide insight into the upper "speed limit" for protein folding rates. The rate constants exhibit a power-law dependence on the number of peptide bonds between the heme and Ru complex. Adherence of our data to a slope of −1.5 is consistent with theoretical models for ideal, freely-jointed Gaussian chain polymers, but its magnitude is smaller than reported for synthetic polypeptides. We also examine rates of contact formation within a stable loop in a His63-heme ligated form of the protein. Additionally, we resolve millisecond-timescale folding by coupling time-resolved fluorescence energy transfer (trFRET) to a continuous flow microfluidic mixer to obtain intramolecular distance distributions throughout the folding process. Our results suggest that cytochrome cb562 is minimally frustrated.