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 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.
Fetrow
J S
JS
Department of Biological Sciences, Center for Biochemistry and Biophysical Chemistry, State University of New York at Albany, Albany, New York 12222, USA. jacque@isadora.albany.edu
Spitzer
J S
JS
Gilden
B M
BM
Mellender
S J
SJ
Begley
T J
TJ
Haas
B J
BJ
Boose
T L
TL
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D045304:Cytochromes c; D005057:Eukaryotic Cells; D005998:Glycine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c; D011392:Proline; D005998:Glycine