Temperature and driving force dependence of the folding rate of reduced horse heart cytochrome c.
Utilizing the stability difference between the ferro and ferri forms of horse heart cytochrome c (cyt c), folding of reduced cyt c was triggered by laser-induced reduction of unfolded oxidized cyt c. Measurements were made of the kinetics of the main folding phase (1 ms-10 s) in which collapsed reduced cyt c transforms to the native conformation. The folding rates were studied extensively as a function of temperature (5-75 degrees C) and guanidine hydrochloride (GdnHCl) concentration (1.6-4.9 M). At constant [GdnHCl], the Arrhenius plot of the folding rate constant (k) is nonlinear. At temperatures above 40 degrees C, the decrease in protein stability counteracts the expected increase in folding rate. Introducing free energy (DeltaG), derived from protein stability data, into the Eyring and Arrhenius equations leads to: ln k = ln(k(b)T/h) + DeltaS()/R - DeltaH()/RT - theta(m)DeltaG/RT = ln A - E(a)/RT - theta(m)DeltaG/RT, where theta(m) is the ratio between the denaturant dependence of the folding rate and the stability. By using this equation at constant DeltaG [or constant equilibrium constant (K)], linear Arrhenius plots are obtained. For the main folding phase of reduced cyt c, a positive DeltaS() is obtained indicating that the transition state is less ordered than the reactant. A model is proposed in which reduced cyt c first collapses into a compact intermediate, which needs to expand to reach the transition state of the rate-limiting folding reaction.
Pascher
T
T
Department of Chemical Physics, Lund University, Getingevägen 60, S-221 00 Lund, Sweden. Torbjorn.Pascher@chemphys.lu.se
D000818:Animals; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability; D019791:Guanidine; D006736:Horses; D007700:Kinetics; D008432:Mathematical Computing; D008956:Models, Chemical; D009206:Myocardium / Q000201:enzymology*; D017711:Nonlinear Dynamics; D010084:Oxidation-Reduction; D010782:Photolysis; D011489:Protein Denaturation; D017510:Protein Folding; D013696:Temperature; D013816:Thermodynamics
D003574:Cytochrome c Group; D019791:Guanidine