title: Tertiary interactions between the fifth and sixth transmembrane segments of rhodopsin.
abstract: We have used cysteine scanning mutagenesis and disulfide cross-linking in a split rhodopsin construct to investigate the secondary structure and tertiary contacts of the fifth (TM5) and sixth (TM6) transmembrane segments of rhodopsin. Using a simple increase in pH to promote disulfide bond formation, three cross-links between residues on the extracellular side of TM5 (at positions 198, 200, and 204) and TM6 (at position 276) have been identified and characterized. The helical pattern of cross-linking observed indicates that the fifth transmembrane helix extends through residue 200 but does not include residue 198. Rhodopsin mutants containing these disulfides demonstrate nativelike absorption spectra and light-dependent activation of transducin, suggesting that large movements on the extracellular side of TM5 with respect to TM6 are not required for receptor activation.
authors.affiliation: Department of Biochemistry, Volen Center for Complex Systems, Brandeis University, Waltham, Massachusetts 02454, USA.
authors.forename: M
authors.initials: M
authors.lastname: Struthers
authors.forename: H
authors.initials: H
authors.lastname: Yu
authors.forename: M
authors.initials: M
authors.lastname: Kono
authors.forename: D D
authors.initials: DD
authors.lastname: Oprian
mesh_terms: D000042:Absorption; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics; D017434:Protein Structure, Tertiary; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism; D013053:Spectrophotometry; D015293:Transducin / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D010446:Peptide Fragments; D010649:Phenylalanine; D012243:Rhodopsin; D015293:Transducin; D003545:Cysteine