Structural constraints imposed by a non-native disulfide cause reversible changes in rhodopsin photointermediate kinetics.
Suspensions of bovine rhodopsin in 2% lauryl maltoside detergent were treated with Cu(phen)(3)(2+) to form a disulfide bridge between cysteines 140 and 222 which occur naturally in the bovine rhodopsin sequence. Absorption difference spectra were collected after excitation with a pulse of 477 nm light on the time scale from 1 micros to 690 ms, and the results were analyzed using global exponential fitting. Only two exponentials could be fit to data from the Cu(phen)(3)(2+)-treated rhodopsin, while three exponentials were needed to fit data either from untreated rhodopsin or from Cu(phen)(3)(2+)-oxidized rhodopsin after further dithiothreitol reduction. Dithiothreitol treatment of rhodopsin which had not been previously oxidized with Cu(phen)(3)(2+) had no effect on the observed kinetics. Since the 140-222 disulfide has previously been shown to block transducin activation, its effects on rhodopsin activation are of considerable interest. Cu(phen)(3)(2+) treatment favors formation of the meta I(380) intermediate relative to meta I(480) and slows formation of meta II from meta I(380). This suggests that the protein change involved in meta I(380) formation is similar to the structural constraint introduced by the 140-222 disulfide. These results show that formation of disulfides in rhodopsin has potential as a tool for discriminating between the three isochromic, 380 nm absorbing intermediates involved in rhodopsin activation and for gaining insight into how their structures differ.
Lewis
J W
JW
Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.
Szundi
I
I
Kliger
D S
DS
D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D007700:Kinetics; D010618:Phenanthrolines / Q000737:chemistry; D010777:Photochemistry; D012243:Rhodopsin / Q000737:chemistry*; D012374:Rod Cell Outer Segment / Q000737:chemistry
D003432:Cross-Linking Reagents; D004220:Disulfides; D010618:Phenanthrolines; C014741:bis(1,10-phenanthroline)copper(2+) ion; D012243:Rhodopsin