Structural features and light-dependent changes in the sequence 306-322 extending from helix VII to the palmitoylation sites in rhodopsin: a site-directed spin-labeling study

Biochemistry. 1999 Jun 22;38(25):7931-7. doi: 10.1021/bi9900121.


Sixteen single-cysteine substitution mutants of rhodopsin were prepared in the sequence 306-321 which begins in transmembrane helix VII and ends at the palmitoylation sites at 322C and 323C. The substituted cysteine residues were modified with a selective reagent to generate a nitroxide side chain, and the electron paramagnetic resonance spectrum of each spin-labeled mutant was analyzed in terms of residue accessibility and mobility. The periodic behavior of these parameters along the sequence indicated that residues 306-314 were in a regular alpha-helical conformation representing the end of helix VII. This helix apparently extends about 1.5 turns above the surface of the membrane, with one face in strong tertiary interaction with the core of the protein. For the segment 315-321, substituted cysteine residues at 317, 318, 320, and 321 had low reactivity with the spin-label reagent. This segment has the most extensive tertiary interactions yet observed in the rhodopsin extra-membrane sequences at the cytoplasmic surface. Previous studies showed the spontaneous formation of a disulfide bond between cysteine residues at 65 and 316. This result indicates that at least some of the tertiary contacts made in the 315-321 segment are with the sequence connecting transmembrane helices I and II. Photoactivation of rhodopsin produces changes in structure detected by spin labels at 306, 313, and 316. The changes at 313 can be accounted for by movements in the adjacent helix VI.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites / genetics
  • Cattle
  • Edetic Acid / analogs & derivatives
  • Electron Spin Resonance Spectroscopy
  • Glucosides
  • Light*
  • Micelles
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed*
  • Palmitic Acid / chemistry*
  • Palmitic Acid / metabolism
  • Peptide Fragments / chemistry*
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Structure, Secondary
  • Rhodopsin / chemistry*
  • Rhodopsin / genetics
  • Rhodopsin / metabolism
  • Spin Labels*
  • Structure-Activity Relationship


  • Glucosides
  • Micelles
  • Peptide Fragments
  • Spin Labels
  • Palmitic Acid
  • EDDA
  • dodecyl maltoside
  • Rhodopsin
  • Edetic Acid