G Protein-Coupled Receptor Rhodopsin: A Prospectus

Supplementary Data

• Figure 1. Drawing of the vertebrate rod photoreceptor cell. This is a highly differentiated post-mitotic cell composed of a sac of internal membranes, called rod outer segment, containing disks, enveloped by the plasma membranes. The ROS is connected to the inner segment by a tiny cilium. This fragile connection allows the ROS to be easily separated and purified by density flotation methods. The most abundant protein of ROS is rhodopsin (> 90%). Bovine retina was a source of highly purified rhodopsin for the crystallographic studies. The rod cell is highly sensitive and can detect a single photon, but saturates at low light intensity (≈10⁴ photons/s). To support such high sensitivity, rhodopsin molecules (10⁸) must be inactive. The spontaneous thermal activation of rhodopsin occurs with a half life of ≈ 400 years. This property implies that rhodopsin must be constrained in the inactive conformation by multiple mechanisms, each of low probability. Once photoactivated, the 11-cis-retinylidene chromophore isomerizes to all-trans-retinylidene and causes propagation and amplification of the signal, activating hundreds of transducin molecules. Download larger version.

  

  ---

  Figure 2. Structure of the main oligosaccharide chains of bovine rhodopsin (6). Mannose moieties are in blue, N-glucosamine moieties are in black, and red denotes the Asn residue. Download larger version.

  

  ---

  Figure 3. Chemical structure and photoisomerization of retinals. (A) Photoisomerization of 11-cis-retinal by the "Hula-Twist" process (56) largely preserves the position of the β-ionone ring and the Schiff base for both isomers at the expense of the polyene chain position. The next stage of the process is imagined to be a "bicycle" twist of the 10-11 and 14-15 single bonds. (B) Superimposed chemical structure of 11-cis-retinal and all-trans-retinal generated by the Hula-Twist photoisomerization process. (C) Superimposed chemical structure of all-trans-retinal and 11-cis-retinal generated by the conventional one-bond-flip photoisomerization process. Note repositioning of β-ionone ring. Download larger version.

  

  ---

  Figure 4. Different views of the rhodopsin dimer in the crystal lattice. Lighter to darker colors denote cytoplasmic (extradiscal) to extracellular (intradiscal) parts of rhodopsin. Oligosaccharide groups are shown in orange. (A) Blue and orange (including oligosaccharide groups) indicate residues that form contacts with other dimers; red notes hydrophobic residues that form contacts between two rhodopsin molecules, including palmitoyl groups. (B,C) Red indicates only palmitoylated residues from both molecules of the dimer. Oligosaccharide groups are shown in orange. (C) The two molecules of (B) are rotated 90º about the vertical axis between the molecules. Download larger version.

  

  ---

  Figure 5. Helices of rhodopsin. Colors denoting residues in contact with the surface (violet), upper charged part of membrane (orange), lower part of membranes (red), and internal residues of rhodopsin (gray). Retinal and Lys²⁹⁶ shown as stick and ball model. (A) View from intradiscal space (extracellular). (B) View from extradiscal space (cytoplasmic). Download larger version.

  

  ---

  Figure 6a. Structure of rhodopsin represented by helical wheels with interhelical contacts. The contact between two residues (or between the chromophore's Schiff base and Glu¹¹³) is defined as any interatomic distance between atoms in the residues smaller than 4.0 Å, slightly larger than the sum of the van der Waals radii. Heavy lines represent ionic/hydrogen interactions. The drawing is similar to one previously published for another GPCR (167). (A) Represents all interactions within rhodopsin. (B) Highlighted residues indicate the positions of mutations associated with retinitis pigmentosa (RP) within the transmembrane segment of rhodopsin. All mutations associated with RP were previously presented in a two-dimensional model (34). Download larger version.

  

  ---

  Figure 6b. Comparison of helices of rhodopsin as determined from the crystal structure (27, 28) and helix traces from frog rhodopsin (gray balls) determined from cryo-electron microscopy (130). (A) View perpendicular to helices. (B) View parallel to main axis of rhodopsin. Download larger version.

  

  ---

  Figure 7. Comparison of helices of rhodopsin as determined from the crystal structure (27, 28) and helix traces from frog rhodopsin (gray balls) determined from cryo-electron microscopy (130). (A) View perpendicular to helices. (B) View parallel to main axis of rhodopsin. Download larger version.

  

  ---

  Figure 8. Top view of rhodopsin with 11-cis-retinylidene and all-trans-retinylidene. (A), The 11-cis-retinylidene (dark violet) and all-trans-retinylidene (light blue) as envisioned in Meta II based on the work of Nakanishi and colleagues (168). Helices I--VIII are colored as a spectrum of visible light from blue (helix I) to red (helix VIII). (B) Probable conformations of retinal during activation of rhodopsin: 11-cis-retinylidene, green; in early photoproducts, all-trans-retinylidene, yellow; and in Meta II, orange. Download larger version.

  

  ---

  Figure 9. Crevices and cavities at the intracellular side and around the chromophore of rhodopsin. (A) Most of the cavities are located in the intracellular part of the protein. Colors denote the depth of the cavity: from blue (surface) to red (inside). The central red cavity contains 11-cis-retinal. (B) Intracellular parts of rhodopsin are lighter in color. Palmitoyl chains are in red, oligosaccharides are in orange. (C) Cavities shown as solid, rhodopsin as a transparent surface. Colors denote the depth of this part of the cavity: from blue (surface) to red (inside). (D) View along main axis of rhodopsin. Retinal is shown without cavity. Calculations of cavities were done with the PASS program (168). All figures with protein structures were prepared in InsightII v.2000, Accelrys Inc. Download larger version.