Chicken apocytochrome c has been shown to possess a much stronger tendency to fold spontaneously in aqueous solution than the equivalent enzyme from other species. In the present work, the amino acid that determines 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 apoprotein and S103A exhibited the same folding property during dialysis renaturation processes as that chemically prepared from chicken cytochrome c, while those containing V92A mutation did not. Quantitative studies by 2,2,2-trifluoroethanol (TFE) and sodium perchlorate (NaClO4) titration demonstrated that the V92A mutation decreased the helix content that could be induced and confirmed that valine 92 is the major determinant of the folding propensity of chicken apocytochrome c. Furthermore, CD spectra, turbidity measurements, and a translocation assay on a model membrane system showed that the V92A mutation also drastically altered the conformation of apocytochrome c after being incorporated into lipid bilayer and decreased the aggregation of phospholipid vesicles after association of the apoprotein, thus rendering the molecule more competent for translocation across the membrane. Our results showed that a single amino acid substitution could radically alter the folding propensity of an unfolded polypeptide chain and thus influence the conformation following its insertion into phospholipid bilayer. Study holds ProTherm entries: 4670, 4671, 4672 Extra Details: folding ability; dialysis renaturation; helix content;,model membrane system; phospholipid bilayer
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:26 p.m.
|Number of data points||5|
|Assays/Quantities/Protocols||Experimental Assay: dG_H2O ; Derived Quantity: ddG_H2O|
|Libraries||Mutations for sequence MGDIEKGKKIFVQKCSQCHTVEKGGKHKTGPNLHGLFGRKTGQAEGFSYTDANKNKGITWGEDTLMEYLENPKKYIPGTKMIFAGIKKKSERVDLIAYLKDATSK|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|