Structure, Function, and Disease Mechanisms in Rhodopsin Phil Reeves (PI) Current Group Members: Dr. Chikwado Opefi (RP Fighting Blindness, RA) Kumars Riyahi (MPhD candidate)

Rhodopsin – dim light photoreceptor of rods ~80 million rhodopsin receptors/rod cell Palczewski, K. (2006) Annu. Rev. Biochem. 75, 743-767

Rhodopsin Light activated G-Protein Coupled Receptor (GPCR). Located in disc membranes of photoreceptor cells. Mediates vision in dim light. A light-sensitive retinylidene ligand is covalently bound. Dark-state and light- activated state structures solved. So what next? light Retinal isomerization GT GDP GTP Vision

Key questions remaining now that innactive and active-state structures of rhodopsin (& other GPCRS) have been solved What are the precise details of receptor activation and G-protein engagement/coupling? Are X-ray structures sufficient? Relevance to general activation of GPCRs? What is the mechanism of ligand exit and entry during the visual cycle – how is retinal isomer selectivity achieved ? Other GPCRs with hydrophobic ligands? How do point mutations in rhodopsin give rise to Retinitis Pigmentosa? How does rhodopsin fold and how does it reach its appropriate subcellular destination?

What techniques do we use and what expertise do we have? We rely heavily on site directed mutagenesis to make mutants that disrupt normal receptor function. Mutant receptors are produced by robust mammalian expression systems and then purified by immunoaffinity chromatography. Transient (10s of μg) or inducible stable cell line based (10s of mg) expression systems are available. We have 10L mammalian bioreactor capability. We have expertise in handling membrane proteins.

Project 1: structure and activation Collaboration with Steve Smith at Stony Brook (SUNY). Construction of mutants, stable cell lines, all biochemical measurements performed at Essex. Large scale expression, stable- isotope labelling, MAS SS NMR measurements performed at Stony Brook. Labelled retinals synthesized by Mudi Sheves (Weizmann Inst.) H4 H3 H5 H7 H2 H1 H6 dark H6 H4 H3 H5 H7 H2 H1 active

Project 2: Retinitis Pigmentosa Retinitis Pigmentosa is an inherited disease that results in blindness. Over 100 mutations map to the RHO gene – most affect folding and stability of rod opsin. We use biochemistry and biophysics to probe misfolding and we try to understand misfolding and stability by repairing the defects. Proteomics.

Project 3: Ligand exit and entry How does all trans retinal leave as the active state decays? How is its re-entry prevented? How is 11-cis retinal selected for uptake? The entry mechanism of hydrophobic ligands must be very different to that used by water soluble ligands that enter at the extracellular surface of most GPCRs. 11-cis All trans

Project 4: role of group conserved residues Small group conserved residues (G, A, S, C, T)

Existing collaborations: Essex - Chris Reynolds (computation, modelling) Metodi Metodiev (proteomics) Philippe Laissue (imaging) Mike Hough (crystallography) UK - Mike Cheetham (UCL) molecular chaperones Belgium - Nico Callaewaert (VIB Ghent) glycobiology USA - Steve Smith (Stony Brook) solid state nmr How might we move forward? Folding kinetics, ligand binding kinetics (other GPCRs), medically relevant mutations in other receptors e.g. cancer, ageing , other disease etc. Our broad strategy is to use disease as a route and justification to study biological function in membrane proteins.