An opsin mutant with increased thermal stability.
This report describes the biochemical characterization of a double mutant of rhodopsin (N2C,D282C) in which Cys residues engineered into the protein at positions 2 (in the amino-terminal extracellular domain) and 282 (in the extracellular loop between transmembrane helices 6 and 7) are shown to form a disulfide bond and increase the thermal stability of the unliganded or opsin form of the protein. Wild-type opsin does not survive detergent solubilization and purification at pH 7.5 and 25 degrees C. In contrast, the N2C,D282C mutant opsin survives the purification protocol and loses less than 50% activity after incubation for 20 days under the same conditions. Less than 5% is lost after 20 days at 4 degrees C. While the disulfide bond clearly has a dramatic effect on protein stability, it has a minor impact on the activity of the pigment. The MII lifetime of the mutant (6.6 min) is similar to that of the wild type (7.9 min), and the specific activity of the light-activated mutant for activation of transducin is within 20% of the wild-type activity. Therefore, it seems likely that the disulfide bond does not perturb greatly the structure of the protein. For these reasons, we anticipate that the mutant may be of use in detailed kinetic and mechanistic investigations of the ligand binding reaction and for crystallization trials involving recombinant rhodopsin, especially the unliganded opsin form of the protein.
Xie
Guifu
G
Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454, USA.
Gross
Alecia K
AK
Oprian
Daniel D
DD
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D019556:COS Cells; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D008027:Light; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012172:Retinaldehyde / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D013816:Thermodynamics; D015293:Transducin / Q000378:metabolism; D014162:Transfection
D003432:Cross-Linking Reagents; D004220:Disulfides; D011994:Recombinant Proteins; D001224:Aspartic Acid; D001216:Asparagine; D012243:Rhodopsin; D015293:Transducin; D003545:Cysteine; D012172:Retinaldehyde; D004229:Dithiothreitol