Guanidine hydrochloride induced unfolding of yeast iso-2 cytochrome c

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Abstract
The properties of the guanidine hydrochloride induced unfolding transition of iso-2 cytochrome c (iso-2) from S. cerevisiae were investigated by using kinetic and equilibrium techniques were compared with previously published studies of horse cytochrome c, which differs from iso-2 by 46% in amino acid sequence. Measurements of absorbance in the UV and visible spectral regions as a function of guanidine hydrochloride concentration give superimposable equilibrium transition curves with a midpoint of 1.15 M at pH 7.2 and 20.degree. C. A 2-state analysis of the equilibrium data gives a Gibbs free energy of unfolding of 3.1 kcal/mol at 20.degree. C in the absence of denaturant. This agrees well with the predicted difference in stability between S. cerevisiae iso-2 and horse cytochrome c estimated from the free energies of transfer of buried hydrophobic groups. Three kinetic phases associated with folding can be detected throughout most of the transision zone. Two of the phases are detected by stopped-flow mixing experiments. The 3rd phase is over within the mixing time of the flow experiments but is detectable by temperature jumps. At 20.degree. C, pH 7.2, the slowest phase (.tau.1) is in the 20-100 s time range, the middle phase (.tau.2) is in the 0.1-3 s range, and the fastest phase (.tau.3) is on the order of 1 ms. For the reactions observed in the stopped flow (.tau.1) and (.tau.2), a simplified 3-state mechanism can be used to predict quantitatively the relative amplitudes of the phases and the equilibrium unfolding curve from the observed time constant data. Previously this same mechanism has been successful in describing the folding reactions of horse cytochrome c. The qualitative features of protein folding reactions may be conserved among homologous proteins.