Site specific combinations of stabilizing and destabilizing amino acid replacements in yeast cytochrome c: in vivo and in vitro effects.
Oligonucleotide-directed mutagenesis in vivo was used to create destabilizing site specific changes at position 6 in iso-1-cytochrome c in Saccharomyces cerevisiae. These changes were made in combination with the stabilizing N52I substitution. The resulting proteins showed that a variety of forces and interactions are responsible for these destabilizations. Increasing side chain size was the strongest predictor of decreases in cytochrome c levels in vivo. With intermediate size replacements, increasing hydrophobicity correlated with the proteins' thermostability. Some differences in protein levels in vivo could not be explained by side chain size and hydrophobicity alone. Therefore, specific interactions of individual amino acids may also be involved. The N52I-stabilizing mutation tended to increase the protein levels to the same degree relative to the amino acid at position 6. These stabilized cytochromes had an increased specific activity when compared to the series with the original N52. Strains with these altered cytochromes c showed temperature sensitivities for protein levels and function. Thermodynamic measurements in vivo of the WT (C102A), N52I, G6A, G6A N52I, and G6S N52I correlated with the in vivo data. The variant G6A N52I showed additivity (Wells, 1990) of the Cm's and delta delta G's of unfolding for guanidine hydrochloride denaturation.
Linske-O'Connell
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Department of Biochemistry, University of Rochester School of Medicine and Dentistry, New York 14642, USA.
Sherman
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McLendon
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D045304:Cytochromes c; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013816:Thermodynamics
D000596:Amino Acids; C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c