Thermodynamic stabilities of yeast iso-1-cytochromes c having amino acid substitutions for lysine 32.
Iso-1-cytochromes c having lysine 32 replaced by leucine, glutamine, tyrosine, and tryptophan were prepared from strains of bakers' yeast, Saccharomyces cerevisiae, and chemically blocked at cysteine 107 with methyl methanethiolsulfonate to prevent dimerization. These modified ferricytochromes c were guanidine denatured, and the unfolding thermodynamics were determined by circular dichroism and fluorescence measurements. Thermal unfolding was also monitored by absorbance measurements. The guanidine denaturation midpoints for the altered proteins are smaller than the wild type, while the orders of stability from unfolding free energy changes are: Lys-32 (wild type) approximately Leu-32 approximately Gln-32 (circular dichroism), greater than Gln-32 (fluorescence) greater than Tyr-32 approximately Trp-32. Midpoints and differences in free energy changes for thermal unfolding parallel the fluorescence free energy changes for guanidine-induced unfolding. Thus, the blocked Leu-32 and Lys-32 proteins are equally stable with respect to both chemical and thermal denaturation. The reported data indicate that single replacements may significantly modify protein stability, and that substitution for an evolutionarily retained residue in normal cytochrome c structures does not always destabilize the protein. In addition, in vitro thermal stabilities approximately correlate with in vivo specific activities.
Hickey
D R
DR
Department of Biochemistry, University of Rochester Medical School, New York 14642.
McLendon
G
G
Sherman
F
F
D003574:Cytochrome c Group / Q000235:genetics / Q000378:metabolism*; D045304:Cytochromes c; D004795:Enzyme Stability; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013053:Spectrophotometry; D013816:Thermodynamics
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D006146:Guanidines; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c; D019791:Guanidine; D008239:Lysine