Conformational dynamics of a fast folding cytochrome captured by electron transfer, time-resolved fluorescence energy transfer, and microfluidic mixing

Cytochrome c-b_(562) belongs to an interesting family of four-helix bundle cytochromes that have nearly identical structures yet widely divergent folding pathways, creating a unique opportunity to study the relationship between primary sequences and folding in proteins with similar topologies. We characterize the dynamics of cytochrome c-b_(562) through a combination of electron transfer and fluorescence energy transfer (FRET). The dynamics of intrachain diffusion in unfolded cytochromes are probed by monitoring contact quenching of a photoexcited, covalently-bound [Ru(bpy)2(IA-phen)]^(2+) dye by the heme Fe^(III). Quenching of the *Ru^(II) luminescence decay provides information about the electron transfer rates, which correspond to the rates of transient contact formation. We resolve fast folding by coupling time-resolved FRET with an ultrafast continuous flow mixer. FRET occurs between a small, covalently-bound dansyl fluorophore and the heme. Fitting the dansyl fluorescence decay following picosecond laser excitation provides us with time-dependent dansyl-heme distance distributions during the two-state folding process