title: Atomistic design of microbial opsin-based blue-shifted optogenetics tools.
abstract: Microbial opsins with a bound chromophore function as photosensitive ion transporters and have been employed in optogenetics for the optical control of neuronal activity. Molecular engineering has been utilized to create colour variants for the functional augmentation of optogenetics tools, but was limited by the complexity of the protein-chromophore interactions. Here we report the development of blue-shifted colour variants by rational design at atomic resolution, achieved through accurate hybrid molecular simulations, electrophysiology and X-ray crystallography. The molecular simulation models and the crystal structure reveal the precisely designed conformational changes of the chromophore induced by combinatory mutations that shrink its π-conjugated system which, together with electrostatic tuning, produce large blue shifts of the absorption spectra by maximally 100 nm, while maintaining photosensitive ion transport activities. The design principle we elaborate is applicable to other microbial opsins, and clarifies the underlying molecular mechanism of the blue-shifted action spectra of microbial opsins recently isolated from natural sources.
authors.affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
authors.forename: Hideaki E
authors.initials: HE
authors.lastname: Kato
authors.affiliation: Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Motoshi
authors.initials: M
authors.lastname: Kamiya
authors.affiliation: Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Seiya
authors.initials: S
authors.lastname: Sugo
authors.affiliation: Department of Bioengineering Sciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Jumpei
authors.initials: J
authors.lastname: Ito
authors.affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
authors.forename: Reiya
authors.initials: R
authors.lastname: Taniguchi
authors.affiliation: Department of Bioengineering Sciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Ayaka
authors.initials: A
authors.lastname: Orito
authors.affiliation: RIKEN SPring-8 Center, Hyogo 679-5148, Japan.
authors.forename: Kunio
authors.initials: K
authors.lastname: Hirata
authors.affiliation: Department of Neuroscience II, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan.
authors.forename: Ayumu
authors.initials: A
authors.lastname: Inutsuka
authors.affiliation: Department of Neuroscience II, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Yamanaka
authors.affiliation: Department of Bioengineering Sciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Andrés D
authors.initials: AD
authors.lastname: Maturana
authors.affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
authors.forename: Ryuichiro
authors.initials: R
authors.lastname: Ishitani
authors.affiliation: Division of Pharmaceutical Sciences, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Sudo
authors.affiliation: Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Shigehiko
authors.initials: S
authors.lastname: Hayashi
authors.affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Nureki
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000378:metabolism; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D004594:Electrophysiology; D004926:Escherichia coli / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007477:Ions; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D009154:Mutation; D009474:Neurons / Q000378:metabolism; D055355:Opsins / Q000737:chemistry*; D062308:Optogenetics / Q000379:methods*; D011487:Protein Conformation; D015202:Protein Engineering; D012243:Rhodopsin / Q000737:chemistry; D017299:Rod Opsins / Q000737:chemistry*; D055672:Static Electricity
chemical_list: D007477:Ions; D055355:Opsins; D017299:Rod Opsins; C095853:short-wavelength opsin; D012243:Rhodopsin