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NMR spectroscopy in studies of light-induced structural changes in mammalian rhodopsin: applicability of solution (19)F NMR - PubMed

  • ️Fri Jan 01 1999

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

J Klein-Seetharaman et al. Proc Natl Acad Sci U S A. 1999.

Abstract

We report high resolution solution (19)F 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), CF(3)-CH(2)-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 degrees C by solution (19)F 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 (19)F 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 (19)F NMR spectroscopy to studies of the tertiary structures in the cytoplasmic face of intact rhodopsin in the dark and on light activation.

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Figures

Figure 1
Figure 1

Six single cysteine mutants of rhodopsin studied in this report, K67C, Cys-140, K245C, K248C, K311C, and Cys-316, are highlighted in a secondary structural model of bovine rhodopsin. For details of their preparation, see text.

Figure 2
Figure 2

A general method for attachment of an 19F label (TET) to the sulfhydryl groups of cysteine residues in the cytoplasmic face of rhodopsin.

Figure 3
Figure 3

(A) Solution 19F NMR spectra in the dark of the single cysteine mutants (Fig. 1). The cysteine residues were derivatized with the TET group as in Fig. 2. All samples contained 0.2 mM TFA, an internal reference, which was set at 0 ppm. Between 400 and 4000 scans were averaged. I, Cys-140 (and small amount of Cys-316); II, Cys-316, (and small amount of Cys-140); III, K311C; IV, K245C; V, K67C; VI, K248C; VII, side products of the labeling procedure (Fig. 2), oxidized TET (TET-TET) and the mixed disulfide (TET-PDS). (B) Dark 19F NMR spectra of TET-labeled Cys-140 rhodopsin in different concentrations of DM, of OG, and in the presence of DMPC. I, DM, 0.1 and 10%; II, OG, 1 and 10%; III, OG 1% and OG (1%) + DMPC (0.15%).

Figure 4
Figure 4

Changes in 19F NMR spectra of TET-labeled cysteine mutants on illumination: red lines, dark spectra; blue lines, spectra after illumination. Each spectrum was taken within 2 min acquisition time (average of 160 scans).

Figure 5
Figure 5

19F NMR spectral changes in the TET-labeled cysteine mutant K248C: in the dark, 3 min after illumination, and at different subsequent time intervals. Acquisition parameters were as in Fig. 4.

Figure 6
Figure 6

Superimposition of the residues studied by 19F NMR on the arrangement of the 7-transmembrane helical bundle based on cryoelectron microscopy studies (5). The electron density contour sections at 13, 15, and 17 Å (gray to black) from the center of the membrane are shown. The contour sections were from a three-dimensional map at an effective resolution 7.5 Å in the membrane plane and 16.5 Å normal to the plane. Residue positions are mapped onto the electron contour sections on the basis of site-directed spin labeling studies (2, 4, 36, 37). The figure has been adapted from Fig. 5 in ref. .

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References

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