Rhodopsin

1. Rhodopsin Christen Eberhart

2. Rhodopsin Sequence

3. The Eye • Rhodopsin is found in the rods that are located in the eye • Rods are composed of stacked disks • Rhodopsin is densely packed into each disk • Rods are responsible for black and white vision • Rhodopsin works best at dim light, responsible for night vision (too much light will saturate the protein) • Rod density is found greater on the outer edges of the retina (peripheral vision)

4. Opsin & Retinal • Rhodopsin is made up of the protein opsin with the chromophore, retinal, covalently attached • The linkage occurs at Lys-296

5. Rhodopsin Structure • 348 amino acids, 40 kD • 7 transmembrane alpha helices connected by six loops of varying lengths • 1 short alpha helix along the cytoplasmic membrane • 2 antiparallel beta sheets at E-II and 2 antiparallel beta sheets at the NH2-terminal • Cys-322 and Cys-323 attach to membrane by palmitic acid residues • Functional monomer, however, can form dimers

6. Retinal • Retinal is a derivative of Vitamin A, which is a derivative of beta-carotene • Isomerization of cis-retinal to trans-retinal by light causes a conformation change in rhodopsin which triggers a signal • Rhodopsin absorbs at green-blue light (500 nm) which makes the protein appear reddish-purple

7. Retinal Binding Site • Lys-296 covalently attaches to retinal by a Schiff base • Glu-113, Gly-114, Ala-117, Thr-118, Gly-120, and Gly-121 side chains make a binding pocket for the retinal • Thr-118, Tyr-268, and Ile-189 possibly could interact with the C9 methyl on the retinylidene group • Met-207, His-211, Phe-212, Tyr-268, and Ala-269 surround the B-ionone ring of the retinal • Tyr-43, Met-44, Leu-47, Thr-94 and Phe-293 region surrounds the Schiff base • Ala-169 may interact with the B-ionone ring in the activated state when retinal is trans

8. Helical Wheel of the Short Alpha Helix • Hydrophobic side chains hold helix parallel to membrane • Hydrophilic side chains provide a key point of contact with the G-protein (Transducin)

9. Stabilization between the Helices • Amino acids that provide stabilization of structure between five helices are shown • Disulfide bond between Cys110 and Cys-187 • Ala-299 peptide carbonyl hydrogen bonds with Asn-55 and Asp-83 • Asp-83 connects by a water molecule to Gly-120 • Asn-78 hydrogen bonds to Ser-127, Thr-160, and Trp-161 • Asn-302 bonds through a water molecule with Asn-83

10. G-protein couple receptor (GPCR) • 7 transmembrane receptor that sense molecules outside the cell and activate inside signal transduction pathways and cellular responses (Rhodopsin) • Found only in eukaryotes • GPCR activates a G-protein (Transducin) by exchanging its GDP for GTP • GPCR is bound to a G-protein while in its inactive state • Once a GPCR is active, the G-protein detaches • G-protein is made up of 3 subunits (Gα, Gβ, and Gγ) • Once G-protein is activated, the Gα subunit activates another protein (phosphodiesterase) and detaches from the other two subunits Rhodopsin Transducin: Gα is red, Gβ is blue, and Gγ is yellow

11. Phototransduction Pathway • Light activates Rhodopsin which activates Transducin by exchanging its GDP for GTP • When active, Transducin’s alpha subunit dissociates from the other two subunits • The active Transducin then activates a membrane bound protein called phosphodiesterase • Phosphodiesterase hydrolyzes cGMP • The hydrolyzes of cGMP, leads to the ion channel closing and the initiation of an action potential • To return back to the inactive state Rhodopsin Kinase phosphorylates the cytosolic tail of rhodopsin which inhibits the activation of transducin • Arrestin then binds to the phosphorylated rhodopsin further inhibiting activity

12. Inactive to Active Form • Active (all trans form of rhodopsin) form still awaits a crystal structure • These pictures are based on computer simulations • Picture on the right: a is the non-active form, b-d is over time changing to the active from • Picture on the bottom shows a potential binding groove opening up in the active form on the cytoplasmic side

13. Multiple Alignment Sequence Red-highly conserved important amino acid Blue-conserved important amino acid Purple-important amino acid Black-highly conserved amino acid Grey-conserved amino acid

14. Retinitis Pigmentosa • Group of genetic eye conditions where the retina of the eye slowly and progressively degenerates • Night blindness occurs early on, then tunnel vision, and then many years later eventually blindness by the age of 40 • Up to 150 different point mutations can lead to RP, which causes misfolding of the protein • The mutant proteins then aggregate together and disrupt the visual phototransduction pathway • Rhodopsin mutations make up 25% cases of RP

15. References • Choi G, et al (2002) Structural studies of metarhodopsin II, the activated form of the G-protein coupled receptor, rhodopsin. Biochemistry, 41(23): 7318-1324. • Berg JM, Tymoczko JL, Stryer L (2002) Biochemistry. New York: W.H. Freeman and Co. • Krisha GA, et al (2002) Evidence that helix 8 of rhodopsin acts as a membrane-dependent conformational switch. Biochemistry, 41 (26): 8298-8309. • Palczewski K, et al (2000) Crystal structure of rhodopsin: A G-protein coupled receptor. Science, Vol. 289, pp. 733-4. • Terakita A, et al (2002) Functional interaction between bovine rhodopsin and G protein transducin. Journal of Biological Chemistry. 277 (1): 40-46. • Helical Wheel (http://kael.net/helical.htm) • Butterfly and Flower (photograph) (http://www.allaboutvision.com) • Magnify (photograph) (http://www.flickr.com/photos/dfrighini/3240329545/)