ANALYSIS OF THE STRUCTURE AND STABILITY OF OMEGA-LOOP-A REPLACEMENTS IN YEAST ISO-1-CYTOCHROME-C

Omega (Omega)-loop A, residues 18-32 in wild-type yeast iso-1-cytochro me c, has been deleted and replaced with loop sequences from three oth er cytochromes c and one from esterase. Yeast expressing a partial loo p deletion do not contain perceptible amounts of holoprotein as measur ed by low-temperature spectroscopy and cannot grow on nonfermentable m edia. Strains expressing loop replacement mutations accumulate holopro tein in vivo, but the protein function Varies depending on the sequenc e and length of the replacement loop; in vivo expression levels do not correlate with their thermal denaturation temperatures. In vitro spec troscopic studies of the loop replacement proteins indicate that all f old into a native-like cytochrome c conformation, but are less stable than the wild-type protein. Decreases in thermal stability are caused by perturbation of loop C backbone in one case and a slight reorganiza tion of the protein hydrophobic core in another case, rather than rear rangement of the loop A backbone. A single-site mutation in one of the replacement mutants designed to relieve inefficient hydrophobic core packing caused by the new loop recovers some, but not all, of the lost stability.