Printed in U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY A Single Amino Acid Substitution

In structure-function studies on bovine rhodopsin by in vitro site-specific mutagenesis, we have prepared three mutants in the cytoplasmic loop between the putative transmembrane helices E and F. In each mu-tant, charged amino acid residues were replaced by neutral residues: mutant 1, Gl~~~’-Gln; mutant 2, LysZ4’+Leu; and mutant 3, G l ~ ~ ~ “ + G l n, LysZ4’dLeu, and G1uZ4’+Gln. The mutant rhodopsin genes were expressed in monkey kidney (COS-1) cells. After the addition of 11-cis-retinal to the cells, the rhodopsin mutants were purified by immunoaffinity adsorption. Each mutant gave a wild-type rhodopsin visible ab-sorption spectrum. The mutants were assayed for their ability to stimulate the GTP- activity of transducin in a light-dependent manner. While mutants 1 and