Jc. Tong et al., V92A MUTATION ALTERED THE FOLDING PROPENSITY OF CHICKEN APOCYTOCHROME-C AND ITS INTERACTION WITH PHOSPHOLIPIDS, Biochemistry, 35(29), 1996, pp. 9460-9468
Chicken apocytochrome c has been shown to possess a much stronger tend
ency to fold spontaneously in aqueous solution than the equivalent enz
yme from other species. In the present work, the amino acid that deter
mines its folding ability was elucidated by site-directed mutagenesis,
Wild-type chicken apocytochrome c and three mutants V92A, S103A, and
V92A/S103A were expressed in Escherichia coli. The wild-type apoprotei
n and S103A exhibited the same folding property during dialysis renatu
ration processes as that chemically prepared from chicken cytochrome c
, while those containing V92A mutation did not. Quantitative studies b
y 2,2,2-trifluoroethanol (TFE) and sodium perchlorate (NaClO4) titrati
on demonstrated that the V92A mutation decreased the helix content tha
t could be induced and confirmed that valine 92 is the major determina
nt of the folding propensity of chicken apocytochrome c. Furthermore,
CD spectra, turbidity measurements, and a translocation assay on a mod
el membrane system showed that the V92A mutation also drastically alte
red the conformation of apocytochrome c after being incorporated into
lipid bilayer and decreased the aggregation of phospholipid vesicles a
fter association of the apoprotein, thus rendering the molecule more c
ompetent for translocation across the membrane. Our results showed tha
t a single amino acid substitution could radically alter the folding p
ropensity of an unfolded polypeptide chain and thus influence the conf
ormation following its insertion into phospholipid bilayer.