title: Fluorescence imaging-based high-throughput screening of fast- and slow-cycling LOV proteins.
abstract: Light-oxygen-voltage (LOV) domains function as blue light-inducible molecular switches. The photosensory LOV domains derived from plants and fungi have provided an indispensable tool for optogenetics. Here we develop a high-throughput screening system to efficiently improve switch-off kinetics of LOV domains. The present system is based on fluorescence imaging of thermal reversion of a flavin cofactor bound to LOV domains. We conducted multi site-directed random mutagenesis of seven amino acid residues surrounding the flavin cofactor of the second LOV domain derived from Avena sativa phototropin 1 (AsLOV2). The gene library was introduced into Escherichia coli cells. Then thermal reversion of AsLOV2 variants, respectively expressed in different bacterial colonies on agar plate, was imaged with a stereoscopic fluorescence microscope. Based on the mutagenesis and imaging-based screening, we isolated 12 different variants showing substantially faster thermal reversion kinetics than wild-type AsLOV2. Among them, AsLOV2-V416T exhibited thermal reversion with a time constant of 2.6 s, 21-fold faster than wild-type AsLOV2. With a slight modification of the present approach, we also have efficiently isolated 8 different decelerated variants, represented by AsLOV2-V416L that exhibited thermal reversion with a time constant of 4.3 × 10(3) s (78-fold slower than wild-type AsLOV2). The present approach based on fluorescence imaging of the thermal reversion of the flavin cofactor is generally applicable to a variety of blue light-inducible molecular switches and may provide a new opportunity for the development of molecular tools for emerging optogenetics.
authors.affiliation: Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo, Japan.
authors.forename: Fuun
authors.initials: F
authors.lastname: Kawano
authors.affiliation: Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Aono
authors.affiliation: Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo, Japan.
authors.forename: Moritoshi
authors.initials: M
authors.lastname: Sato
mesh_terms: D018554:Avena / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry / Q000379:methods*; D056949:Phototropins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D056949:Phototropins