Thermal denaturation of iso-l-cytochrome c variants: Comparison with solvent denaturation

Lynn M. Herrmann, Bruce E. Bowler

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Thermal denaturation studies as a function of pH were carried out on wild-type iso-1-cytochrome c and three variants of this protein at the solvent-exposed position 73 of the sequence. By examining the enthalpy and T(m) at various pH values, the heat capacity increment (ΔC(p)), 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 ΔC(p) value (1.15 ± 0.17 kcal/mol K) decreased slightly relative to wild-type iso-1-cytochrome c (1.40 ± 0.06 kcal/mol K), while for the Ile 73 (1.65 ± 0.07 kcal/mol K) and the Val 73 (1.50 ± 0.06 kcal/mol K) variants, ΔC(p), 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 (Herrmann L, Bowler BE, Dong A, Caughey WS. 1995. The effects of hydrophilic to hydrophobic surface mutations on the denatured state of iso-1-cytochrome c: Investigation of aliphatic residues. Biochemistry 34:3040-3047). Both the m-value and ΔC(p) 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-1-cytochrome c, a lack of correlation exists which implies that there may he 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 m-values and ΔC(p) for these variants will be discussed in this context.

Original languageEnglish
Pages (from-to)657-665
Number of pages9
JournalProtein Science
Issue number3
StatePublished - Mar 1997


  • circular dichroism
  • guanidine hydrochloride denaturation
  • heat capacity increment
  • iso-1-cytochrome c
  • m-values
  • protein stability
  • thermal denaturation


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