STRUCTURE, FUNCTION, AND TEMPERATURE SENSITIVITY OF DIRECTED, RANDOM MUTANTS AT PROLINE-76 AND GLYCINE-77 IN OMEGA-LOOP-D OF YEAST ISO-1-CYTOCHROME-C

Residues 75-78 form a tight turn within Omega-loop D in Saccharomyces cerevisiae iso-1-cytochrome c. Directed, random mutagenesis of invaria nt residues proline 76 and glycine 77 in this rum were analyzed for th e in vivo functionality and level of protein within the cell. All prot eins, except Pro76Val, also exhibit a significant decrease in intracel lular cytochrome c levels, ranging from 15% to 80% of wild type. Furth ermore, all isolated mutant strains, except the one expressing Pro76Va l, exhibit a significant decrease in growth on lactate medium, suggest ing that the variant cytochromes are much less functional than, wild t ype. This requirement for protein function is clearly the cause for th e strict invariance of these residues in eukaryotic cytochromes c. Sev en proteins with mutations just at Pro76 were purified and studied by circular dichroism spectroscopy. All proteins with mutations at Pro76 exhibit melting temperatures about 7 degrees C less than that of the w ild-type protein, suggesting that mutation of Pro76 affects the entrop y of the denatured state. It is proposed that the functional significa nce of Pro76 and Gly77 is the requirement for a type II (beta(gamma L) ) beta-turn in this loop, the conformation of which requires a glycine at the third position, and that a change occurs in this turn conforma tion upon a change in the redox state of the protein.