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.FRET | microfluidic mixing | protein refolding

We have investigated intrachain contact dynamics in unfolded cytochrome cb562 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 timescale 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.

Maltose-binding protein (MBP) is in a molten globule state at pH 3 as characterized by ANS binding. DEER measurements of seven spin-labeled double mutants as in the native state at pH 7 had shown excellent agreement with X-ray data. At pH 3 corresponding DEER measurements of all the mutants yield a broad distribution of distances. This was to be expected if there is no defined tertiary structure and the individual helices pointing into all possible directions. However, as MBP still binds maltose as molten globule although more weakly, the native structure must be retained at or near the active site. This is now being investigated with a new set of mutants.