Solution 19F nuclear Overhauser effects in structural studies of the cytoplasmic domain of mammalian rhodopsin

Abstract

19F nuclear Overhauser effects (NOEs) between fluorine labels on the cytoplasmic domain of rhodopsin solubilized in detergent micelles are reported. Previously, high-resolution solution (19)F NMR spectra of fluorine-labeled rhodopsin in detergent micelles were described, demonstrating the applicability of this technique to studies of tertiary structure in the cytoplasmic domain. To quantitate tertiary contacts we have applied a transient one-dimensional difference NOE solution (19)F NMR experiment to this system, permitting assessment of proximities between fluorine labels specifically incorporated into different regions of the cytoplasmic face. Three dicysteine substitution mutants (Cys-140-Cys-316, Cys-65-Cys-316, and Cys-139-Cys-251) were labeled by attachment of the trifluoroethylthio group through a disulfide linkage. Each mutant rhodopsin was prepared (8-10 mg) in dodecylmaltoside and analyzed at 20 degrees C by solution (19)F NMR. Distinct chemical shifts were observed for all of the rhodopsin (19)F labels in the dark. An up-field shift of the Cys-316 resonance in the Cys-65-Cys-316 mutant suggests a close proximity between the two residues. When analyzed for (19)F-(19)F NOEs, a moderate negative enhancement was observed for the Cys-65-Cys-316 pair and a strong negative enhancement was observed for the Cys-139-Cys-251 pair, indicating proximity between these sites. No NOE enhancement was observed for the Cys-140-Cys-316 pair. These NOE effects demonstrate a solution (19)F NMR method for analysis of tertiary contacts in high molecular weight proteins, including membrane proteins.

Figure 1

Schematic representation of a cysteine side chain attached via a disulfide bond to the ¹⁹F TET label.

Figure 2

WT (A) and two dicysteine rhodopsin mutants (B and C), Cys-65–Cys-316 and Cys-139–Cys-251, are highlighted in secondary structural models of bovine rhodopsin. WT cysteines and mutations to cysteine residues are highlighted with circles. Deleted WT cysteines, replaced with serine, are highlighted with squares.

Figure 3

Solution ¹⁹F NMR spectra in the dark of WT and the two dicysteine mutants. The cysteine residues were derivatized with the TET group as described in the text. All samples contained 0.2 mM TFA as an internal reference, which was set to 0.0 ppm. Approximately 2,000 scans were averaged per spectrum. (A) WT Cys-140–Cys-316. (B) Cys-65–Cys-316. (C) Cys-139–Cys-251.

Figure 4

(A) Selective ¹⁹F inversion experiment on Cys-65–Cys-316 by the progressive inversion-recovery method (17). Shown is the recovery of the 9.2-ppm resonance line in the Cys-65–Cys-316 1D-¹⁹F solution NMR spectrum after selective inversion by a 180^o pulse. The relaxation delay times (t) were (i) 0.0, (ii) 0.02, (iii) 0.04, (iv) 0.06, (v) 0.1, and (vi) 1.5 s. (B) Control spectra for Cys-65–Cys-316 collected at identical values of t as in A, after selective irradiation of a blank region (show with a bar) of the NMR spectrum. (C) Difference spectra for Cys-65–Cys-316 resulting from subtraction of control spectra (as shown in B) from inverted spectra (as shown in A). The NOE enhancement at 9.7 ppm is marked with an arrow. Approximately 2,000 scans were averaged for each spectrum in A and B.

Figure 5

NOE enhancements are plotted against t compared with WT (●) for (A) Cys-65–Cys-316 (■) and (B) Cys-139–Cys-251 (▴). Each point is the average of three measurements. The error given is the SEM.