EPR-Detected Folding Kinetics of Externally Located Cysteine-Directed Spin-Labeled Mutants of Iso-1-cytochrome c

We report the application of our newly developed dielectric resonator-based flow and stopped-flow kinetic EPR systematically to probe protein folding in yeast iso-1-cytochrome c at cysteine-directed spin-labeled locations. The locations studied have not been previously directly probed by other techniques, and we observe them on a time scale stretching from 50 μs to seconds. On the basis of crystal structure and homology information, the following mutation-tolerant, externally located cysteine labeling sites were chosen (in helices, T8C, E66C, and N92C; in loops, E21C, V28C, H39C, D50C, and K79C), and labeling at these sites was not destabilizing. Dilution of denaturant was used to induce folding and thereby to cause a change in the spin label EPR signal as folding altered the motion of the spin label. Under folding conditions, including the presence of imidazole to eliminate kinetic trapping due to heme misligation, a phase of folding on the 20−30 ms time scale was found. This phase occurred not only at the T8C and N92C labeling sites in the N- and C-terminal helices, where such a phase has been associated with folding in these helices, but overall at labeling sites throughout the protein. In the absence of imidazole the 20−30 ms phase disappeared, and another phase having the time scale of 1 s appeared throughout the protein. There was evidence under all conditions for a burst phase on a scale of less than several milliseconds which occurred at labeling positions V28C, H39C, D50C, E66C, and K79C in the middle of the protein sequence. At spin-labeled D50C rapid-mix flow EPR indicated a very short ∼50 μs phase possibly associated with the prefolding or compaction of the loop to which D50 belongs. Spin labels have been criticized as perturbing the phenomena which they measure, but our spin labeling strategy has reported common kinetic themes and not perturbed, disconnected kinetic events.