NMR spectroscopy in studies of light-induced structural changes in mammalian rhodopsin: Applicability of solution 19F NMR

Judith Klein-Seetharaman, Elena V. Getmanova, Michèle C. Loewen, Philip J. Reeves, H. Gobind Khorana

Research output: Contribution to journalArticlepeer-review

96 Scopus citations

Abstract

We report high resolution solution 19F NMR spectra of fluorine-labeled rhodopsin mutants in detergent micelles. Single cysteine substitution mutants in the cytoplasmic face of rhodopsin were labeled by attachment of the trifluoroethylthio (TET), CF3-CH2-S, group through a disulfide linkage. TET-labeled cysteine mutants at amino acid positions 67, 140, 245, 248, 311, and 316 in rhodopsin were thus prepared. Purified mutant rhodopsins (6-10 mg), in dodecylmaltoside, were analyzed at 20°C by solution 19F NMR spectroscopy. The spectra recorded in the dark showed the following chemical shifts relative to trifluoroacetate: Cys-67, 9.8 ppm; Cys-140, 10.6 ppm; Cys-245, 9.9 ppm; Cys-248, 9.5 ppm; Cys-311, 9.9 ppm; and Cys-316, 10.0 ppm. Thus, all mutants showed chemical shifts downfield that of free TET (6.5 ppm). On illumination to form metarhodopsin II, upfield changes in chemical shift were observed for 19F labels at positions 67 (-0.2 ppm) and 140 (-0.4 ppm) and downfield changes for positions 248 (+0.1 ppm) and 316 (+0.1 ppm) whereas little or no change was observed at positions 311 and 245. On decay of metarhodopsin II, the chemical shifts reverted largely to those originally observed in the dark. The results demonstrate the applicability of solution 19F NMR spectroscopy to studies of the tertiary structures in the cytoplasmic face of intact rhodopsin in the dark and on light activation.

Original languageEnglish (US)
Pages (from-to)13744-13749
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume96
Issue number24
DOIs
StatePublished - Nov 23 1999
Externally publishedYes

Keywords

  • Conformational change
  • G-protein-coupled receptors
  • Membrane proteins
  • Signal transduction
  • Site-directed F labeling

ASJC Scopus subject areas

  • General

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