Chimeric Proton-Pumping Rhodopsins Containing the Cytoplasmic Loop of Bovine Rhodopsin

et al. (2014) Chimeric Proton-Pumping Rhodopsins Containing the Cytoplasmic Loop of Bovine Rhodopsin. PLoS ONE 9(3): e91323. doi:10.1371/journal.pone.0091323 Chimeric Proton-Pumping Rhodopsins Containing the Cytoplasmic Loop of Bovine Rhodopsin Hideki Kandori 0 Kengo Sasaki 0 Takahiro Yamashita 0 Kazuho Yoshida 0 Keiichi Inoue 0 Yoshinori Shichida 0 Karl-Wilhelm Koch, University of Oldenburg, Germany 0 1 Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya, Japan, 2 Department of Biophysics, Graduate School of Science, Kyoto University , Kyoto, Japan, 3 PRESTO , Japan Science and Technology Agency , Honcho Kawaguchi, Saitama , Japan G-protein-coupled receptors (GPCRs) transmit stimuli to intracellular signaling systems. Rhodopsin (Rh), which is a prototypical GPCR, possesses an 11-cis retinal. Photoisomerization of 11-cis to all-trans leads to structural changes in the protein of cytoplasmic loops, activating G-protein. Microbial rhodopsins are similar heptahelical membrane proteins that function as bacterial sensors, light-driven ion-pumps, or light-gated channels. They possess an all-trans retinal, and photoisomerization to 13-cis triggers structural changes in protein. Despite these similarities, there is no sequence homology between visual and microbial rhodopsins, and microbial rhodopsins do not activate G-proteins. In this study, new chimeric proton-pumping rhodopsins, proteorhodopsin (PR) and Gloeobacter rhodopsin (GR) were designed by replacing cytoplasmic loops with bovine Rh loops. Although G-protein was not activated by the PR chimeras, all 12 GR chimeras activated G-protein. The GR chimera containing the second cytoplasmic loop of bovine Rh did not activate G-protein. However, the chimera with a second and third double-loop further enhanced G-protein activation. Introduction of an E132Q mutation slowed the photocycle 30-fold and enhanced activation. The highest catalytic activity of the GR chimera was still 3,200 times lower than bovine Rh but only 64 times lower than amphioxus Go-rhodopsin. This GR chimera showed a strong absorption change of the amide-I band on a light-minus-dark difference FTIR spectrum which could represent a larger helical opening, important for G-protein activation. The light-dependent catalytic activity of this GR chimera makes it a potential optogenetic tool for enzymatic activation by light. - Funding: This work was financially supported by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology to TY (24115509), KI (24115508, 24655009), YS (25251036), and HK (22247024, 25620011). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Rhodopsin (Rh) is one of the G protein-coupled receptors (GPCRs) that has diverged into a photoreceptive protein in retinal visual cells [16]. It is a membrane protein consisting of a single polypeptide opsin and a light-absorbing chromophore 11-cisretinal. Opsin contains seven transmembrane a-helices, which form a structural motif typical of GPCRs. An 11 cis retinal is bound to Lys-296 in the transmembrane helix 7 through a protonated Schiff base linkage. Absorption of a photon by the chromophore causes isomerization to the all-trans form, followed by conformational changes to the protein [1,3]. Several intermediate states in the bleaching process have been identified as photorhodopsin, bathorhodopsin (Batho), lumirhodopsin (Lumi), metarhodopsin-I (Meta-I), and metarhodopsin-II (Meta-II). MetaII catalyzes the GDP-GTP exchange reaction in the transduction of trimeric G protein (Gt) [1,7,8]. Some archaea and bacteria possess retinal binding proteins, which are microbial rhodopsins that contain an all-trans retinal as a chromophore. Among them, the most studied is bacteriorhodopsin (BR), which is found in Halobacterium salinarum [3,911]. This archaea contains four retinal-bonding proteins: BR, halorhodopsin (HR), sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII; also called phoborhodopsin) [12]. BR and HR are light-driven ion pumps, acting as an outward proton- and an inward chloridepump, respectively [9,1214]. SRI and SRII, which are photoreceptors of this halophilic archaea, act in attractant and repellent responses, respectively during phototaxis [15,16]. There are no sequence homologies between visual and microbial rhodopsins, though both possess similar chromophore (retinal) and protein (7-transmembrane helices) structures. It is generally believed that both evolved independently, although the most recent study suggests possibility convergent evolution between both types of rhodopsins [17].) In fact, visual rhodopsins do not transport ions, while microbial rhodopsins do not activate G-proteins. However, Geiser et al. reported that BR chimeras containing the third cytoplasmic loop of bovine Rh are able to activate G-protein [18]. Although the activation level was reported to be similar between native bovine Rh and the chimera (BR/ Rh223-253), we recently quantified the activation level to be 0.003% of bovine Rh [19]. In addition, we reported that chimeras of Natronomonas pharaonis SRII containing third loops of bovine Rh are able to activate G-protein [19]. This observation suggests that a common structural feature for light-induced activation among BR, SRII and bovine Rh exists in which the helix opening motion on the cytoplasmic side probably exposes the third loop to possible binding with G-protein. These chimeras have the potential to be tools for optogenetics [20,21]. In fact, Arian et al. developed a versatile family of genetically encoded optical tools (optoXRs), in which a bovine Rh chimera containing the cytoplasmic loop of other GPCRs was used to respond to light [22]. Even though Rh may be a promising optogenetic tool, it has no photocycle, but bleaches upon light absorption [6]. In addition, 11-cis retinal, the chromophore molecule of Rh, is not generally abundant in animal cells. This limits the practical application of optoXRs. In contrast, our chimera containing an all-trans retinal exhibited a photocycle without any bleaching and was being repeatedly applicable [19]. Endogenous all-trans retinal is sufficient for optogenetics in animal cells [14,23]. Thus, our chimeras can be tested for light-induced enzymatic activation in optogenetics. The greatest problem of these chimeras is likely their low activation level (0.003% of bovine Rh) [19]. In this study, chimeric proton-pumping rhodopsins were designed using Proteorhodopsin (PR) from marine c-proteobacteria [24] and Gloeobacter rhodopsin (GR) from thylakoidless cyanobacteria to make new microbial rhodopsin chimeras with a higher G-protein activating function [25,26]. Experimental Procedures Sample Preparation Chimera constructs were designed based on the wild-type (WT) PR (triple cysteine mutant, TCM (...truncated)