PAPER www.rsc.org/pps | Photochemical & Photobiological Sciences Functional interaction structures of the photochromic retinal protein rhodopsin † Kristina Kirchberg, Tai-Yang Kim, Sebastian Haase and Ulrike Alexiev* Received 16th October 2009, Accepted 27th November 2009 First published as an Advance Article on the web 15th January 2010 DOI: 10.1039/b9pp00134d We studied functional interaction structures of the vertebrate membrane photoreceptor rhodopsin containing retinal as a chromophore. Using time-resolved fluorescence depolarization we analyzed real-time dynamics and conformational changes of the cytoplasmic helix 8 (H8) preceding the long C-terminal tail of rhodopsin. H8 runs parallel to the membrane surface and extends from transmembrane helix 7 whose highly conserved NPxxY(x)F motif connects that region of rhodopsin with the retinal binding pocket. Our measurements indicate that photo-induced retinal isomerization from 11-cis to all-trans provokes conformational changes of H8, including slower motion and reduced flexibility, that are specific for the active metarhodopsin-II photo-intermediate. These conformational changes are absent in the retinal-devoid state opsin and in the phosphorylated metarhodopsin-II state upon receptor deactivation. Furthermore we show that membrane rim effects can influence interfacial reactions at the cytoplasmic rhodopsin surface such as proton transfer reactions between surface and aqueous bulk phase or binding of the signaling protein transducin visualized with single-molecule widefield microscopy. These findings are important for an understanding of the effects of membrane structure on the photo-transduction mechanism. Introduction Photochromic proteins play a key role in energy conversion and visual perception. Rhodopsins belong to the family of seven-transmembrane helix proteins which carry retinal as a chromophore that is covalently linked to the apo-protein via a protonated Schiff base (PSB).‡ Upon excitation by light, the initial event in microbial rhodopsins is retinal isomerization from all-trans to 13-cis. 1,2 This event triggers a cyclic reaction which involves Schiff base (SB) deprotonation, proton transfer steps, conformational changes of the protein, and the reversal of retinal isomerization. Ion pumps, like bacteriorhodopsin and halorhodopsin, 3,4 and light-gated ion channels (channel-rhodopsins) that control photomovement of microalgae 5-7 are prominent members of microbial rhodopsins. In vertebrate retinal photoreceptors, visual rhodopsin molecules are densely packed in the membranes of distinct disk-like or- ganelles (disk) stacked in the outer segment of the rod cell. Rhodopsin is the primary molecule involved in the signaling process that eventually converts a single photon into a visual Freie Universit¨ at Berlin, Inst. f¨ ur. Experimentalphysik, Arnimallee 14 D-14195 Berlin, , Germany. E-mail: alexiev@physik.fu-berlin.de; Fax: +49- 30-83856510; Tel: +49-30-83855157 † This paper is part of a themed issue on synthetic and natural photo- switches. ‡The abbreviations used are: SB, Schiff base; PSB, protonated Schiff base; TM1-7, transmembrane helix 1-7; bathorhodopsin, lumirhodopsin, metarhodopsin-I, and metarhodopsin-II, photoproducts of rhodopsin; MII, metarhodopsin-II; H8, helix 8; Rho, rhodopsin; G T , heterotrimeric G-protein transducin; Arr, arrestin; PDE, phosphodiesterase; PDE*, activated phosphodiesterase; GTP, guanosine-5¢-triphosphate; GDP, guanosine-5¢-diphosphate; GMP, guanosine-5¢-monophosphate; cGMP, cyclic guanosine monophosphate; P, phosphate; P-rhodopsin, phosphory- lated rhodopsin. response. The dense stacking of the disks in the rod outer segments ensures a high probability of single photon absorption. 8 The chromophoric group of visual rhodopsin consists of 11-cis retinal covalently bound to a conserved lysine residue (K296 in bovine rhodopsin) in transmembrane helix (TM) 7 and interacting amino acid residues in the retinal binding pocket that influence the absorption properties of the chromophoric group and control the photochemical pathways of the retinal (Fig. 1A). Glutamic acid in position 113 (E113) in TM3 acts as the primary counter-ion of the PSB 9 and this salt-bridge maintains the inactive conformation of the rhodopsin dark state. Photo-activation leads to retinal isomerization from 11-cis to all-trans on the femtosecond time scale and deprotonation of the nitrogen in the SB group in the transition from the metarhodopsin-I to the metarhodopsin- II intermediate with internal proton transfer to E113. 10-14 These events initiate conformational and H-bonding changes in the receptor as well as proton uptake by rhodopsin. This results within milliseconds in the formation of the active photoprod- uct metarhodopsin-II, which is capable of interacting via its extramembranous cytoplasmic loops with the signaling protein transducin (G T ), a member of the G-protein superfamily (Fig. 1B, 1C). The cytoplasmic TM3/TM6 network (Fig. 1A), including glutamic acid in position 134 (E134) of the conserved E(D)RY motif at the cytoplasmic end of TM3, is supposed to be involved in proton uptake. Protonation of E134 in metarhodopsin-II from the solvent has been shown recently by FTIR spectroscopy. 15 Further interhelical networks, including TM1, TM2 and TM7 as well as TM3 and TM5 contain the carboxylic acids D83 (TM2) and E122 (TM3) in distal and dorsal parts of the retinal binding pocket (Fig. 1A). Both of these residues undergo changes in their hydrogen bonding properties upon photo-activation. D83 connects via a H-bonding network the chromophore binding 226 | Photochem. Photobiol. Sci., 2010, 9, 226–233 This journal is © The Royal Society of Chemistry and Owner Societies 2010