To study the role of omega loop D, residues 70 -84, in the structure and function of yeast iso-1-cytochrome c, this loop was replaced with homologous and heterologous loops. A novel method was developed for rapid insertion of these mutations into the yeast chromosome at the CYC1 locus. The strains containing these loop replacement cytochromes cannot grow on nonfermentable carbon sources, indicating that the proteins are nonfunctional. Whole cell difference spectroscopy shows that no holocytochrome c is present; however, apoprotein is found by immunoblot analysis. Thus, apoprotein is present in these mutant strains, but it cannot bind heme and cannot compete with wild type apoprotein conversion to holoprotein. This is a unique example of a set of loop replacements that do not produce folded protein, and these results suggest that the loop D amino acid sequence in iso-1-cytochrome c plays a significant role in cytochrome c biosynthesis in vivo. To identify the significant amino acids in loop D, random mutagenesis of six highly conserved loop residues, Tyr-74, Ile-75, Pro-76, Gly-77, Thr-78, and Lys-79, was accomplished. Sequencing of the random mutants shows that strict conservation of none of these residues is required to produce a minimally functional cytochrome c. Preferences are found for small, hydrophilic or aromatic residues at position 74, hydrophobic residues at position 75, glycine and arginine at positions 76 and 77, and ␤-branched amino acids at position 78. Implications for the role of loop D in the structure and function of iso-1-cytochrome c are discussed.The protein folding problem is an important, unsolved problem in molecular biology. An understanding of this problem is important to utilization of the data being generated by the human genome project and to further the design and engineering of proteins with specific structures and functions. Loops and turns, the connections between the regular secondary structures, are critical to the design of properly folded proteins. These structures are difficult to characterize because of their range of conformations. The short ␤-turns and reverse turns were the first to be characterized (1, 2); then the longer ⍀-loops were described (3); and finally a more comprehensive definition of loops was published (Ref. 4; for review, see Ref. 5). We are interested in defining the role that loops, particularly ⍀-loops, play in protein folding, function, and stability.⍀-Loops are nonregular protein secondary structures defined as a sequence of six or more residues with a short distance between segment termini and a lack of regular hydrogen bonding between backbone residues (3). However, significant hydrogen bonding and hydrophobic interactions can exist between side chain and backbone atoms within the loop itself.

Residues 75-78 form a tight turn within Omega-loop D in Saccharomyces cerevisiae iso-1-cytochrome c. Directed, random mutagenesis of invariant residues proline 76 and glycine 77 in this turn were analyzed for the in vivo functionality and level of protein within the cell. All proteins, except Pro76Val, also exhibit a significant decrease in intracellular cytochrome c levels, ranging from 15% to 80% of wild type. Furthermore, all isolated mutant strains, except the one expressing Pro76Val, exhibit a significant decrease in growth on lactate medium, suggesting that the variant cytochromes are much less functional than wild type. This requirement for protein function is clearly the cause for the strict invariance of these residues in eukaryotic cytochromes c. Seven 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 degreesC less than that of the wild-type protein, suggesting that mutation of Pro76 affects the entropy of the denatured state. It is proposed that the functional significance of Pro76 and Gly77 is the requirement for a type II (betagammaL) beta-turn in this loop, the conformation of which requires a glycine at the third position, and that a change occurs in this turn conformation upon a change in the redox state of the protein.