title: Chimeric proton-pumping rhodopsins containing the cytoplasmic loop of bovine rhodopsin.
abstract: 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.
authors.affiliation: Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya, Japan.
authors.forename: Kengo
authors.initials: K
authors.lastname: Sasaki
authors.affiliation: Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Yamashita
authors.affiliation: Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya, Japan.
authors.forename: Kazuho
authors.initials: K
authors.lastname: Yoshida
authors.affiliation: Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya, Japan; PRESTO, Japan Science and Technology Agency, Honcho Kawaguchi, Saitama, Japan.
authors.forename: Keiichi
authors.initials: K
authors.lastname: Inoue
authors.affiliation: Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Shichida
authors.affiliation: Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Kandori
mesh_terms: D065638:Absorption, Radiation; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D000458:Cyanobacteria; D003593:Cytoplasm; D008969:Molecular Sequence Data; D055668:Photochemical Processes; D015202:Protein Engineering; D017494:Proton Pumps / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025604:Rhodopsins, Microbial
chemical_list: D001426:Bacterial Proteins; D017494:Proton Pumps; D011993:Recombinant Fusion Proteins; D025604:Rhodopsins, Microbial; C413548:proteorhodopsin; D012243:Rhodopsin