Measuring denatured state energetics: Deviations from random coil behaviorand implications for the folding of iso-1-cytochrome c

The changes in the free energy of the denatured state of a set of yeast iso -1-cytochrome c variants with single surface histidine residues have been m easured in 3 M guanidine hydrochloride. The thermodynamics of unfolding by guanidine hydrochloride is also reported. All variants have decreased stabi lity relative to the wild-type protein. The free energy of the denatured st ate was determined in 3 M guanidine hydrochloride by evaluating the strengt h of heme-histidine ligation through determination of the pK(a) for loss of histidine binding to the heme. The data are corrected for the presence of the N-terminal amino group which also ligates to the heme under similar sol ution conditions. Significant deviations from random coil behavior are obse rved. Relative to a variant with a single histidine at position 26, residua l structure of the order of -1.0 to -2.5 kcal/mol is seen for the other var iants studied. The data explain the slower folding of yeast iso-l-cytochrom e c relative to the horse protein. The greater number of histidines and the greater strength of ligation are expected to slow conversion of the histid ine-misligated forms to the obligatory aquo-heme intermediate during the li gand exchange phase of folding. The particularly strong association of hist idine residues at positions 54 and 89 may indicate regions of the protein w ith strong energetic propensities to collapse against the heme during early folding events, consistent with available data in the literature on early folding events for cytochrome c. (C) 2000 Academic Press.