Magic angle spinning NMR of the protonated retinylidene Schiff base nitrogen in rhodopsin: Expression of 15N-lysine- and 13C-glycine-labeled opsin in a stable cell line (HEK293S cellsyG protein-coupled receptorysignal transductiony11-cis retinalyvisual pigment)

The apoprotein corresponding to the mammalian photoreceptor rhodopsin has been expressed by using suspension cultures of HEK293S cells in defined media that contained 6-15N-lysine and 2-13C-glycine. Typical yields were 1.5–1.8 mgyliter. Incorporation of 6-15N-lysine was quantitative, whereas that of 2-13C-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 15N resonance corresponding to the protonated retinylidene Schiff base nitrogen was observed at 156.8 ppm in the MAS spectrum of 6-15N-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 years. A number of MAS NMR approaches have been developed for measuring the magnitudes and orientations of chemical shift and dipolar interactions in membrane systems (9, 10). These measurements can be directly related to internuclear distances and torsion angles. The advantages of MAS NMR are considerable. Membrane proteins can be studied in their native membrane environment, reaction intermediates can be trapped at low temperature, and structural measurements are possible with ultra-high resolution. Internuclear distances can be measured with resolution on the order of 0.2 Å (11), while torsion angles can be determined to within 610° (12). Such high-resolution measurements are well suited for establishing the key helix–retinal and helix–helix interactions that may be involved in receptor activation. High-level expression of the rhodopsin gene in a suspensionadapted HEK293 stable cell line recently has allowed the preparation of milligram amounts of rhodopsin (13). By using defined media containing 6-15N-lysine and 2-13C-glycine, we now report that milligram quantities of isotopically labeled rhodopsin can be obtained in a similar fashion. This ability to incorporate isotope labels into rhodopsin on a large scale overcomes the major limitation for NMR structural measurements and opens the door for studies of this diverse and important G protein-coupled receptor family. We present MAS NMR spectra of rhodopsin containing isotopically labeled amino acids. Incorporation of 6-15N-lysine into rhodopsin allows us to uniquely target the PSB bond that links the retinylidene chromophore to Lys-296 on transmembrane helix 7. The interaction between the PSB and the Glu-113 counterion is of considerable interest because of its critical role in spectral tuning (14, 15) and receptor activation (16). The 15N chemical shift at 156.8 ppm now reported is characteristic of a PSB nitrogen that has a weak counterion interaction. A long effective Schiff base–counterion distance suggested by the 15N chemical shift is consistent with a structural water molecule in the retinal binding site bridging the counterion and the Schiff base proton (17, 18).§ MATERIALS AND METHODS Material. The sources of all reagents for cell culture and rhodopsin purification have been described (13) except for the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. PNAS is available online at www.pnas.org. Abbreviations: DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; FBS, fetal bovine serum; MAS, magic angle spinning; PSB, protonated Schiff base. ‡To whom reprint requests should be addressed. §This is paper no. 28 in the series ‘‘Structure and Function in Rhodopsin.’’ Paper no. 27 is ref. 8.