Snapshots of a protein folding intermediate

We have investigated the folding dynamics of Thermus thermophilus cytochrome c_(552) by time-resolved fluorescence energy transfer between the heme and each of seven site-specific fluorescent probes. We have found both an equilibrium unfolding intermediate and a distinct refolding intermediate from kinetics studies. Depending on the protein region monitored, we observed either two-state or three-state denaturation transitions. The unfolding intermediate associated with three-state folding exhibited native contacts in β-sheet and C-terminal helix regions. We probed the formation of a refolding intermediate by time-resolved fluorescence energy transfer between residue 110 and the heme using a continuous flow mixer. The intermediate ensemble, a heterogeneous mixture of compact and extended polypeptides, forms in a millisecond, substantially slower than the ∼100-μs formation of a burst-phase intermediate in cytochrome c. The surprising finding is that, unlike for cytochrome c, there is an observable folding intermediate, but no microsecond burst phase in the folding kinetics of the structurally related thermostable protein

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

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

Intrachain Contact Dynamics in Unfolded Cytochrome <i>cb</i>₅₆₂

We have investigated intrachain contact dynamics in unfolded cytochrome <i>cb</i>₅₆₂ by monitoring heme quenching of excited ruthenium photosensitizers covalently bound to residues along the polypeptide. Intrachain diffusion for chemically denatured proteins proceeds on the microsecond time scale with an upper limit of 0.1 μs. The rate constants exhibit a power-law dependence on the number of peptide bonds between the heme and Ru complex. The power-law exponent of −1.5 is consistent with theoretical models for freely jointed Gaussian chains, but its magnitude is smaller than that reported for several synthetic polypeptides. Contact formation within a stable loop was examined in a His63-heme ligated form of the protein under denaturing conditions. Loop formation accelerated contact kinetics for the Ru66 labeling site, owing to reduction in the length of the peptide separating redox sites. For other labeling sites within the stable loop, quenching rates were modestly reduced compared to the open chain polymer