NMR Spectroscopy of Phosphorylated Wild-Type Rhodopsin: Mobility of the Phosphorylated C-Terminus of Rhodopsin in the Dark and upon Light Activation

Elena Getmanova, Ashish B. Patel, Judith Klein-Seetharaman, Michele C. Loewen, Philip J. Reeves, Noga Friedman, Mordechai Sheves, Steven O. Smith, H. Gobind Khorana

Research output: Contribution to journalArticlepeer-review

35 Scopus citations

Abstract

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 31P 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 °C. Below this temperature, the 31P resonance broadened and was only observable using cross polarization. These results indicate that the phosphorylated C-terminus is highly mobile above -40 °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, 19F 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 19F and the 31P-labels. The REDOR technique allows 19F⋯31P distances to be measured out to ∼12 Å 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 19F sites on helix 8 (Cys 316) and in the second cytoplasmic loop (Cys140) are greater than 12 Å in phosphorylated rhodopsin.

Original languageEnglish (US)
Pages (from-to)1126-1133
Number of pages8
JournalBiochemistry
Volume43
Issue number4
DOIs
StatePublished - Feb 3 2004
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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