Combined solid state and solution NMR studies of alpha,epsilon-15N labeled bovine rhodopsin.
Rhodopsin is the visual pigment of the vertebrate rod photoreceptor cell and is the only member of the G protein coupled receptor family for which a crystal structure is available. Towards the study of dynamics in rhodopsin, we report NMR-spectroscopic investigations of alpha,epsilon-15N-tryptophan labeled rhodopsin in detergent micelles and reconstituted in phospholipids. Using a combination of solid state 13C,15N-REDOR and HETCOR experiments of all possible 13C'(i-1) carbonyl/15N(i)-tryptophan isotope labeled amide pairs, and H/D exchange 1H,15N-HSQC experiments conducted in solution, we assigned chemical shifts to all five rhodopsin tryptophan backbone 15N nuclei and partially to their bound protons. 1H,15N chemical shift assignment was achieved for indole side chains of Trp35(1.30) and Trp175(4.65). 15N chemical shifts were found to be similar when comparing those obtained in the native like reconstituted lipid environment and those obtained in detergent micelles for all tryptophans except Trp175(4.65) at the membrane interface. The results suggest that the integrated solution and solid state NMR approach presented provides highly complementary information in the study of structure and dynamics of large membrane proteins like rhodopsin.
Werner
Karla
K
Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-Universität Frankfurt, Max-von-Laue-Str. 7, 60438 Frankfurt/Main, Germany.
Lehner
Ines
I
Dhiman
Harpreet Kaur
HK
Richter
Christian
C
Glaubitz
Clemens
C
Schwalbe
Harald
H
Klein-Seetharaman
Judith
J
Khorana
H Gobind
HG
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000737:chemistry*; D012996:Solutions / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry
D009587:Nitrogen Isotopes; D012996:Solutions; D014364:Tryptophan; D012243:Rhodopsin