Kinetics of Intermolecular Interaction during Protein Folding of Reduced Cytochrome c

Kinetics of intermolecular interaction between reduced cytochrome c (Cyt c) protein and solvent during the protein-refolding process is studied by monitoring the time dependence of apparent diffusion coefficient ( D) using the pulsed-laser-induced transient grating technique. The refolding was triggered by photoinduced reduction of unfolded Fe(III) Cyt c in 3.5 M guanidine hydrochloride (GdnHCl) solution and the change in the diffusion coefficient was monitored in time domain. The relationship between D and the protein conformations under equilibrium condition were investigated at various GdnHCl concentrations using a photolabeling reagent. The time dependence of the observed transient grating signal was analyzed using these data and two models: a continuous change model of the intermolecular interaction and a two-state model. It was found that the TG signals in various time ranges can be consistently reproduced well by the two-state model. The dynamics of D is expressed well by a single exponential function with a rate constant of 22 ± 7 s −1 in a whole time range. The folding process of Cyt c is discussed based on these observations.