Changing the transition state for protein (Un) folding.
(Un)folding transition states of Saccharomyces cerevisiae iso-1-ferri- and ferrocytochromes c were studied using equilibrium and kinetic denaturation experiments. The wild-type protein and the global suppressor variant, N52I (isoleucine replaces asparagine 52), were examined. Denaturation was induced by guanidinium chloride (GdmCI) and monitored by circular dichroism (CD) spectropolarimetry without stopped-flow devices. Soret CD spectra indicate that thermal and GdmCl denatured states are different, and heat is the more effective denaturant. Equilibrium data show that the high stability of ferrocytochrome c can be rationalized as a requirement to bury the oxidation-induced positive charge and remain folded under physiological conditions. Kinetic data are monoexponential and permit characterization of the rate-limiting transition state for unfolding as a function of [GdmCl]. For the oxidized wild-type protein, the transition state solvent accessibility is nearly the same as that of the denatured state. Three perturbations, reducing the wild-type protein, reducing the N52I variant, and substituting position 52 in the oxidized protein, change the free energy and solvent accessibility of the transition state. In contrast, substituting position 52 in the reduced protein apparently does not change the transition state solvent accessibility, allowing more detailed characterization. In the reduced proteins' transition states at 4.3 M GdmCl, the position 52 side chain is in a denatured environment, even though transition state solvent accessibility is only one-third that of the denatured state (relative to the native state).
Doyle
D F
DF
Department of Chemistry, University of North Carolina, Chapel Hill 27599, USA.
Waldner
J C
JC
Parikh
S
S
Alcazar-Roman
L
L
Pielak
G J
GJ
D001216:Asparagine; D002151:Calorimetry; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry*; D045304:Cytochromes c; D014644:Genetic Variation; D019791:Guanidine; D006146:Guanidines; D007532:Isoleucine; D007700:Kinetics; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012680:Sensitivity and Specificity; D013816:Thermodynamics
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D006146:Guanidines; D029701:Saccharomyces cerevisiae Proteins; D007532:Isoleucine; D001216:Asparagine; D045304:Cytochromes c; D019791:Guanidine