Two-state folding of horse ferrocytochrome c: analyses of linear free energy relationship, chevron curvature, and stopped-flow burst relaxation kinetics.
Ferrocytchrome c is a classic two-state fast folder. This assurance comes from extensive equilibrium and kinetic folding studies carried out under strictly anaerobic conditions at 22 degrees C. Conventional guanidine hydrochloride (GdnHCl)-induced unfolding transitions monitored by the use of a sizable set of optical probes do not reveal the accumulation of any intermediate to a detectable level. The GdnHCl dependence of unfolding free energy (DeltaG(D)) is linear over the full range of the denaturant concentration. The GdnHCl folding chevron is characterized by curvatures in both folding and unfolding limbs. However, refolding rates as a function of urea in the presence of different concentrations of GdnHCl yield m(++)f values (the kinetic m-value) that are quantitatively identical. This result, analyzed in terms of the denaturant dependence of the difference in the extent of solvent exposure between a relatively fixed transition state and the preceding state involved in the transition, suggests that the chevron curvature is not related to differential accumulation of a folding intermediate with varying concentration of GdnHCl in the refolding medium. Denaturant dependence of stopped-flow burst signals recorded in normal refolding experiments (pH 7, 22 degrees C) is essentially identical with that recorded in simulating experiments in which the protein stays steadily unfolded even in the denaturant-diluted medium (pH 1.5-2, 22 or 43 degrees C depending on the use of urea or GdnHCl), and they match the denaturant dependence of equilibrium signals for the unfolded protein. The results demonstrate that the burst phase does not entail an early folding intermediate. Rather, the folding kinetics are essentially two-state. These results are central to the phenomenological description of protein folding.
Kumar
Rajesh
R
School of Chemistry, University of Hyderabad, Hyderabad 500046, India.
Bhuyan
Abani K
AK
D000818:Animals; D045304:Cytochromes c / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D019791:Guanidine / Q000494:pharmacology; D006736:Horses; D007202:Indicators and Reagents; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
D007202:Indicators and Reagents; D014508:Urea; D045304:Cytochromes c; D019791:Guanidine