-cytochrome c variants: Comparison with solvent denaturation

Thermal denaturation studies as a function of pH were carried out on wild-type iso-I-cytochrome c and three variants of this protein at the solvent-exposed position 73 of the sequence. By examining the enthalpy and T, at various pH values, the heat capacity increment (AC,,), which is dominated by the degree of change in nonpolar hydration upon protein unfolding, was found for the wild type where lysine 73 is normally present and for three variants. For the Trp 73 variant, the ACp value (1.15 k 0.17 kcal/mol K) decreased slightly relative to wild-type isoI-cytochrome c (1.40 f 0.06 kcal/mol K), while for the Ile 73 (1.65 k 0.07 kcal/mol K) and the Val 73 ( 1 S O k 0.06 kcal/mol K) variants, ACp increased slightly. In previous studies, the Trp 73, Ile 73, and Val 73 variants have been shown to have decreased m-values in guanidine hydrochloride denaturations relative to the wild-type protein (Henmann L, Bowler BE, Dong A, Caughey WS. 1995. The effects of hydrophilic to hydrophobic surface mutations on the denatured state of iso-lcytochrome c: Investigation of aliphatic residues. Biochemistry 343040-3047). Both the m-value and AC,, are related to the change in solvent exposure upon unfolding and other investigators have shown a correlation exists between these two parameters. However, for this subset of variants of iso-I-cytochrome c, a lack of correlation exists which implies that there may be basic differences between the guanidine hydrochloride and thermal denaturations of this protein. Spectroscopic data are consistent with different denatured states for thermal and guanidine hydrochloride unfolding. The different response of rn-values and ACp for these variants will be discussed in this context.