PCR based mutagenesis will be used to selectively alter the amino acid sequence of wild type horse cytochrome c and a variant, H33N, which lacks a non-native histidine ligand of the heme iron and thus exhibits a simplified folding mechanism. Twelve evolutionarily conserved sites in the structural core of the protein, which are unlikely to contribute to function, have been identified for mutagenesis. Mutant proteins will be expressed using a yeast strain which lacks cytochrome c. Recombinant protein purified by NaCI-ethyl acetate precipitation, Amberlite CG-50 chromatography, ultrafiltration and FPLC, will be examined for solubility, stability, and folding - unfolding equilibrium. Tryptophan fluorescence will assess structural stability. This method provides an indication of the overall dimensions of cytochrome c since the single tryptophan in the polypeptide is fluorescent in the unfolded state but loses fluorescence in the native state as a result of energy transfer to heme. Far UV CD spectroscopy will monitor the equilibria of folding and unfolding under various conditions of denaturation and temperature.