Abstract
Rhodopsin contains two cysteines (Cys110 and Cys187) that are highly conserved among members of the G protein coupled receptor family and that form a disulfide bond connecting helixes 3 and 4 on the extracellular side of the protein. However, recent work on a rhodopsin mutant split in the cytoplasmic loop connecting helixes 3 and 4 has shown that the amino- and carboxy-terminal fragments of this split protein do not comigrate on nonreducing SDS−PAGE gels, suggesting that the native Cys110−Cys187 disulfide bond is not present in this mutant [Ridge et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 3204−3208; Yu et al. (1995) Biochemistry 34, 14963−14969]. We show here that the inability to observe the disulfide bond on SDS gels is the result of a disulfide bond exchange reaction which occurs when this split rhodopsin is denatured in preparation for SDS−PAGE. Cys185 reacts with the native disulfide, displacing Cys110 and forming a new disulfide with Cys187. If the sulfhydryl-specific reagent N-ethylmaleimide is included in the sample during preparation for electrophoresis or if Cys185 is changed to Ser, the two fragments do comigrate with full-length rhodopsin on SDS gels and, therefore, are connected by the native Cys110−Cys187 disulfide bond. In related experiments, we find no evidence that the Cys110−Cys187 disulfide bond is broken upon formation of the active intermediate metarhodopsin II.