Tertiary Interactions between the Fifth and Sixth Transmembrane Segments of Rhodopsin

• Published in: Biochemistry (Easton) Vol. 38; no. 20; pp. 6597 - 6603
• Main Authors: Struthers, Mary, Yu, Hongbo, Kono, Masahiro, Oprian, Daniel D.
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 18.05.1999
• Subjects: Absorption; Amino Acid Substitution - genetics; Animals; Cattle; Cell Membrane - chemistry; Cell Membrane - metabolism; Cross-Linking Reagents - chemistry; Cysteine - genetics; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Mutagenesis, Site-Directed; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Phenylalanine - genetics; Protein Structure, Tertiary; Rhodopsin - chemistry; Rhodopsin - metabolism; Spectrophotometry; Transducin - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9902384

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.