Folding/Unfolding Kinetics of Mutant Forms of Iso-1-cytochrome c with Replacement of Proline-71

Proline-71. an evolutionally conserved residue that separates two short α-helical reeons. is replaced by valine, threonine, or isoleucine in at least partially functional forms of iso-1-cytochrome c from Saccharomyces cerevisiae [Ernst, J. F., Hampsey, D. M., Stewart, J. W., Rackovsky, S., Goldstein, D., & Sherman, F. (1985) J. Biol. Chem. 260, 13225-13236]. To assign the effects of perturbations at position 71 to steps in the process of protein folding, the kinetic properties of the folding/unfolding reactions of normal protein and the three mutant forms are compared. At pH 6.0, 20 °C, fluorescence-detected folding/unfolding kinetics are monitored below, within, and above the equilibrium transition zone by using stopped-flow mixing to perform guanidine hydrochloride concentration jumps. Three kinetic phases are detected for each of the four proteins. The fastest of these phases (τ₃) differs in rate for the wild type and mutant proteins. The remaining kinetic phases (τ₁ and τ₂) have similar rates for all four proteins over the entire range of folding/unfolding conditions. The guanidine hydrochloride dependence of the relative amplitudes of the kinetic phases is complex and is sensitive to the nature of the substituent at position 71: each of the four proteins shows differences in the fraction of folding/unfolding associated with the two fastest rate processes. The results suggest that it is the location of the mutation in the primary structure rather than the nature of the substituent that determines which kinetic step (or steps) is changed in rate. However, the kinetic amplitudes (α₂ and α₃) are very sensitive to the nature of the substituent at position 71. Thus different mutations do have different effects on the stability of the species responsible for fast folding phases. Contrary to the expectations of the proline isomerization hypothesis [Brandts, J. F., Halvorson, H. R., & Brennan, M. (1975) Biochemistry 14, 4953–4963], replacements of Pro-71 have little effect on fluorescence-detected slow refolding. Replacement of Pro-71 by Val-71 does not affect either the amplitude (α₁) or time constant (τ₁)for slow refolding. Neither the Thr-71 nor the Ile-71 replacement alters the rate of slow refolding, although decreases in relative amplitude are observed.