Protein folding dynamics of cytochrome c seen by transient grating and transient absorption spectroscopies.

We investigate optically triggered protein folding dynamics of cytochrome c (Cytc) using transient grating (TG) and transient absorption (TA) spectroscopies. Despite many studies on protein folding dynamics of Cytc, a well-known model protein, direct spectroscopic evidence for the three-dimensional global folding process has been rarely reported. By measuring the TG signal of CO-bound Cytc (Cytc-CO) in the presence of a denaturant, we clearly detected the change of diffusion coefficient that reflects the size change of Cytc upon photodissociation of the CO ligand from unfolded Cytc-CO. The quantitative analysis of TG signals supports that the optically triggered folding reaction of Cytc in the presence of a denaturant takes place through a detectable intermediate (three-state folding kinetics). This is in contrast with the two-state folding dynamics of Cytc under a denaturant-free environment without any detectable intermediate. (1) From the quantitative global analysis of the TG signals, the rate constants for the U → I and I → N transitions in a CAPS buffer solution (pH 7) at room temperature in the presence of a denaturant at various concentrations are determined to be 1065 ± 17 to 3476 ± 103 s(-1) and 101 ± 6 to 589 ± 21 s(-1), respectively. In addition, the activation energies (E(a)) for the U → I and I → N transitions are determined to be 8.7 ± 1.0 kcal/mol and 7.1 ± 1.3 kcal/mol, respectively. The folding dynamics of Cytc initiated by the CO photolysis is discussed based in terms of the protein size change.