NMR spectroscopy of phosphorylated wild-type rhodopsin: mobility of the phosphorylated C-terminus of rhodopsin in the dark and upon light activation.
Binding of arrestin to light-activated rhodopsin involves recognition of the phosphorylated C-terminus and several residues on the cytoplasmic surface of the receptor. These sites are in close proximity in dark, unphosphorylated rhodopsin. To address the position and mobility of the phosphorylated C-terminus in the active and inactive receptor, we combined high-resolution solution and solid state NMR spectroscopy of the intact mammalian photoreceptor rhodopsin in detergent micelles as a function of temperature. The (31)P NMR resonance of rhodopsin phosphorylated by rhodopsin kinase at the C-terminal tail was observable with single pulse excitation using magic angle spinning until the sample temperature reached -40 degrees C. Below this temperature, the (31)P resonance broadened and was only observable using cross polarization. These results indicate that the phosphorylated C-terminus is highly mobile above -40 degrees C and immobilized at lower temperature. To probe the relative position of the immobilized phosphorylated C-terminus with respect to the cytoplasmic domain of rhodopsin, (19)F labels were introduced at positions 140 and 316 by the reaction of rhodopsin with 2,2,2-trifluoroethanethiol (TET). Solid state rotational-echo double-resonance (REDOR) NMR was used to probe the internuclear distance between the (19)F and the (31)P-labels. The REDOR technique allows (19)F...(31)P distances to be measured out to approximately 12 A with high resolution, but no significant dephasing was observed in the REDOR experiment in the dark or upon light activation. This result indicates that the distances between the phosphorylated sites on the C-terminus and the (19)F sites on helix 8 (Cys 316) and in the second cytoplasmic loop (Cys140) are greater than 12 A in phosphorylated rhodopsin.
Getmanova
Elena
E
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Patel
Ashish B
AB
Klein-Seetharaman
Judith
J
Loewen
Michele C
MC
Reeves
Philip J
PJ
Friedman
Noga
N
Sheves
Mordechai
M
Smith
Steven O
SO
Khorana
H Gobind
HG
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000737:chemistry; D003624:Darkness; D005461:Fluorine / Q000378:metabolism; D008027:Light; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D010759:Phosphorus Isotopes / Q000378:metabolism; D010766:Phosphorylation; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism*; D012996:Solutions; D014270:Trifluoroethanol / Q000737:chemistry
D010446:Peptide Fragments; D010759:Phosphorus Isotopes; D012996:Solutions; D005461:Fluorine; D014270:Trifluoroethanol; D012243:Rhodopsin; D003545:Cysteine