Bovine rhodopsin

Figure 3.3 Molecular structure of G-protein-coupled receptors. In (a) the electron density map of bovine rhodopsin is shown as obtained by cryoelectron microscopy of two-dimensional arrays of receptors embedded in lipid membrane. The electron densities show seven peaks reflecting the seven a-helices which are predicted to cross the cell membrane. In (b) is shown a helical-wheel diagram of the receptor orientated according to the electron density map shown in (a). The diagram is seen as the receptor would be viewed from outside the cell membrane. The agonist binding pocket is illustrated by the hatched region between TM3, TM5 and TM6. (From Schertler et al. 1993 and Baldwin 1993, reproduced from Schwartz 1996). Reprinted with permission from Textbook of Receptor Pharmacology. Eds Foreman, JC and Johansen, T. Copyright CRC Press, Boca Raton, Florida...

FIGURE 2.1 A side view of the structure of the prototype G-protein-coupled, 7TM receptor rhodopsin. The x-ray structure of bovine rhodopsin is shown with horizontal gray lines, indicating the limits of the cellular lipid membrane. The retinal ligand is shown in a space-filling model as the cloud in the middle of the structure. The seven transmembrane (7TM) helices are shown in solid ribbon form. Note that TM-III is rather tilted (see TM-III at the extracellular and intracellular end of the helix) and that kinks are present in several of the other helices, such as TM-V (to the left), TM-VI (in front of the retinal), and TM-VII. In all of these cases, these kinks are due to the presence of a well-conserved proline residue, which creates a weak point in the helical structure. These kinks are believed to be of functional importance in the activation mechanism for 7TM receptors in general. Also note the amphipathic helix-VIII which is located parallel to the membrane at the membrane interface. 

Ovchinnikov 234 237) has shown that bovine rhodopsin, although quite different in amino acid sequence (348 residues), also forms seven transmembrane helices. This structural similarity between bacterial and mammalian light activated membrane proteins is remarkable. Since the two amino acid sequences have little in common it would appear that the necessary requirement is seven transmembrane helices to form a channel which is specific for proton migration. For example it has been suggested that a similar arrangement and function is performed by the lactose permease of E. coli237). 

Jacobsen, R.B., Sale, K.L., Ayson, M.J., Novak, P., Hong, J., Lane, P., Wood, N.L., Kruppa, G.H., Young, M.M., and Schoeniger, J.S. (2006) Structure and dynamics of dark-state bovine rhodopsin revealed by chemical cross-linking and high-resolution mass spectrometry. Protein Sci. 15, 1303-1317. 

FIGURE 49-3 Proposed transmembrane disposition of bovine rhodopsin. Sugar moieties at asparagine-2 and asparagine-15 are shown with red arrows. Palmitoyl groups at cysteine-322 and cysteine-323 are indicated with broken lines. Hydroxyamino acid residues that may be phosphorylated by rhodopsin kinase are clustered in a C-terminal domain of the molecule. 

Krebs, A., Villa, C., Edwards, P. C., and Schertler, G. F. (1998) Characterisation of an improved two-dimensional p22121 crystal from bovine rhodopsin. J. Mol. Biol. 282, 991-1003. 

Fig. 5.1 Schematic drawing of membrane association modes of peptides A Integral membrane proteins (1) major fd coat protein gpVIII of bacteriophage Ml 3 (pdb lfdm), anchored by an 18-residue trans-membrane hydrophobic helix (2) bovine rhodopsin, a 7 trans-membrane domain (G-protein-coupled) receptor (pdb lf88) (3) ion channel peptaibol Chrysospermin C (pdb lee7), and B Peripheral membrane proteins (1) neuro-...

Fig. 2.162. Absorption spectra of Amphiopl expressed in HEK293s cells (a) and the HPLC patterns of retinal oximes (b). Absorption spectra and the HPLC patterns were measured before (a, curve 1, and b, top trace) and after irradiation at 520 nm for 2 min (a, curve 2, and b, middle trace). The HPLC pattern of retinal oximes extracted from a mixture of irradiated and non-irradiated bovine rhodopsin in equal amounts is indicated as a reference (b, bottom trace). The absorption maxima of the original pigment and its phoroproduct are shown in panel (a). Reprinted with permission from M. Koyanagi et al. [334].

Ovchinnikov, Y., Abdulaev, N., and Bogachuk, A. (1988) Two adjacent cysteine residues in the C-terminal cytoplasmic fragment of bovine rhodopsin are palmitoylated. FEES Lett. 230, 1-5. 

This is an assumption shared with homology modeling based on bovine rhodopsin, which uses as a starting point an inactive form of the receptor and subsequent manipulation is required to transform it into an active form. 

In 1985 Tyminski etal. [55, 56] reported that two-component lipid vesicles of a neutral phospholipid, e.g. DOPC, and a neutral polymerizable PC, bis-DenPC (15), formed stable homogeneous bilayer vesicles prior to photopolymerization. After photopolymerization of a homogeneous 1 1 molar lipid mixture, the lipid vesicles were titrated with bovine rhodopsin-octyl glucoside micelles in a manner that maintained the octyl glucoside concentration below the surfactant critical micelle concentration. Consequently there was insufficient surfactant to keep the membrane protein, rhodopsin, soluble in the aqueous buffer. These conditions favor the insertion of transmembrane proteins into lipid bilayers. After addition and incubation, the bilayer vesicles were purified on a... 

S ATP -I- 338-SKTETSQVAPA-348 <1, 12> (<1, 12> peptide containing the last 11 amino acids of the C-terminal of bovine rhodopsin [20, 24] <1> phosphorylated at Ser-343, about 11% of the rate with rhodopsin, photo-activated rhodopsin-dependent, soluble active kinase catalyzes photoacti-vated rhodopsin-independent peptide phosphorylation [20] <12> only in the presence of photoactivated rhodopsin, which activates RK for peptide phosphorylation, also activated by metarhodopsin III, but not by opsin, up to 60% of the rate with photoactivated rhodopsin, light-dependent phosphorylation [24]) (Reversibility <1,12> [20,24]) [20, 24]... 

Fig. 8.1 Sequence alignment of the four hARs (A, A2a, A, A3), bovine rhodopsin, hp2 adrenergic receptor, and turkey pj adrenergic receptor. In grey are highlighted the transmembrane regions, in red the highly conserved residues and in yellow cysteines that form disulfide linkages that involve EL2. For Ar A2B, AjARs only the cysteine residues that form the conserved disulfide bridge between TM3 and EL2 are highlighted in yellow, because information about other disulfide bonds is not available...

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