State-dependent disulfide cross-linking in rhodopsin.
In previous studies, we developed a new method for detecting tertiary interactions in rhodopsin using split receptors and disulfide cross-linking. Cysteines are engineered into separate fragments of the split opsin, the disulfide bond can be formed between the juxtaposed residues by treatment with Cu(phen)3(2+), and then disulfide cross-links can be detected on the gel by an electrophoretic mobility shift. In this study, we utilized this method to examine the cross-linking reactions between native cysteines in the ground state and after photoexcitation of rhodopsin. In the dark, Cys140 on transmembrane segment (TM) 3 cross-links to Cys222 on TM5. After photobleaching, Cys140 cross-links to Cys316 and Cys222, and the rate of the cross-linking reaction between Cys140 and Cys222 significantly increases.
Yu
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Department of Biochemistry, Volen Center for Complex Systems, Brandeis University, Waltham, Massachusetts 02454, USA.
Kono
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Oprian
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D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003624:Darkness; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010775:Photic Stimulation; D017434:Protein Structure, Tertiary; D012160:Retina; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017299:Rod Opsins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D003432:Cross-Linking Reagents; D004220:Disulfides; D010446:Peptide Fragments; D017299:Rod Opsins; D012243:Rhodopsin; D003545:Cysteine