Magic angle spinning NMR of the protonated retinylidene Schiff base nitrogen in rhodopsin: Expression of ¹⁵ N-lysine- and ¹³ C-glycine-labeled opsin in a stable cell line

Abstract

The apoprotein corresponding to the mammalian photoreceptor rhodopsin has been expressed by using suspension cultures of HEK293S cells in defined media that contained 6-15 N-lysine and 2-13 C-glycine. Typical yields were 1.5-1.8 mg͞liter. Incorporation of 6-15 N-lysine was quantitative, whereas that of 2-13 C-glycine was about 60%. The rhodopsin pigment formed by binding of 11-cis retinal was spectrally indistinguishable from native bovine rhodopsin. Magic angle spinning (MAS) NMR spectra of labeled rhodopsin were obtained after its incorporation into liposomes. The 15 N resonance corresponding to the protonated retinylidene Schiff base nitrogen was observed at 156.8 ppm in the MAS spectrum of 6-15 N-lysine-labeled rhodopsin. This chemical shift corresponds to an effective Schiff base-counterion distance of greater than 4 Å, consistent with structural water in the binding site hydrogen bonded with the Schiff base nitrogen and the Glu-113 counterion. The present study demonstrates that structural studies of rhodopsin and other G protein-coupled receptors by using MAS NMR are feasible.Considerable progress has been made in recent years in understanding structure-function relationships in rhodopsin, the mammalian photoreceptor, and other members of the G protein-coupled receptor family (1-3). These receptors share a common structural motif consisting of seven transmembrane helices ( Fig. 1). Electron diffraction measurements of twodimensional rhodopsin crystals have revealed the spatial arrangement of the seven transmembrane helices (4, 5), whereas a combination of systematic cysteine replacements in the cytoplasmic domain of the receptor followed by spin labeling and EPR measurements has begun to show the nature of the movements in the transmembrane helices that are involved in receptor activation (6-8). An essential feature of the activation mechanism that emerges from these studies is that structural changes in the transmembrane domain are tightly coupled to the conformations of the cytoplasmic and intradiscal (extracellular) loops of the protein.In rhodopsin, receptor activation is controlled by the retinylidene chromophore. The 11-cis isomer of retinal is covalently attached as a protonated Schiff base (PSB) in the interior of the transmembrane domain. Photochemical isomerization of the retinal breaks helix-helix interactions, which, in the dark, lock rhodopsin in the inactive conformation. The idea that receptor-specific helix-helix interactions are involved in activation has arisen in studies on a number of G proteincoupled receptors (GPCRs) (2). Unfortunately, the lack of a high-resolution structure of rhodopsin, or of any other GPCR, has presented a formidable problem for establishing how the transmembrane helices pack and how structural changes in the transmembrane helices are coupled to ligand binding, retinal isomerization, or motion in the other domains of the receptor.Magic angle spinning (MAS) NMR spectroscopy has been increasingly applied to investigate membrane proteins over the past 10 yea...

Vision in the ultraviolet

“…b Bovine rod numbering. NMR [85]. In these latter experiments, the estimated SB to counterion distance of 4.1 and 4.4 Å and the high C = N frequency can be reconciled by the inclusion of a structural water molecule into the retinal binding site ( fig.…”

Location of Trp265 in Metarhodopsin II: Implications for the Activation Mechanism of the Visual Receptor Rhodopsin

“…The suspension growth medium was supplemented with specific 13 C-labeled amino acids (Cambridge Isotope Laboratories, Andover, MA), dialyzed fetal bovine serum (10%), Pluronic F-68 (0.1%), heparin (50 mg/l), penicillin (100 units/ml) and streptomycin (100 mg/ml). 48,50 The cells were induced on day 5 with 2 mg/l tetracycline 46 and harvested on day 7.…”

“…47 Fetal bovine serum (FBS) was dialyzed three times against 20 l of buffer A (137 mM NaCl, 2.7 mM KCl, 1.8 mM KH 2 PO 4 , 10 mM Na 2 HPO 4 , pH 7.2). 48 The cells were grown in Dulbecco's modified Eagle's medium formulation 49 prepared from cell culture-tested components (Sigma, St. Louis, MO). The suspension growth medium was supplemented with specific 13 C-labeled amino acids (Cambridge Isotope Laboratories, Andover, MA), dialyzed fetal bovine serum (10%), Pluronic F-68 (0.1%), heparin (50 mg/l), penicillin (100 units/ml) and streptomycin (100 mg/ml).…”

Changes in Interhelical Hydrogen Bonding upon Rhodopsin Activation

“…44 Media containing [U- 13 45 Fetal bovine serum (500 ml) was dialyzed three times against 20 l of buffer A (137 mM NaCl, 2.7 mM KCl, 1.8 mM KH 2 PO 4 , 10 mM Na 2 HPO 4 (pH 7.2)) as described. 46 Suspension growth and expression of isotopically labeled rhodopsin and mutant rhodopsin were performed as described. [46][47][48] Cells were fed on day 4 with amino acids and ZnCl 2 (Sigma, St Louis, MO) to give a final concentration of 10 mM zinc.…”