title: Overexpression in yeast, photocycle, and in vitro structural change of an avian putative magnetoreceptor cryptochrome4.
abstract: Cryptochromes (CRYs) have been found in a wide variety of living organisms and can function as blue light photoreceptors, circadian clock molecules, or magnetoreceptors. Non-mammalian vertebrates have CRY4 in addition to the CRY1 and CRY2 circadian clock components. Though the function of CRY4 is not well understood, chicken CRY4 (cCRY4) may be a magnetoreceptor because of its high level of expression in the retina and light-dependent structural changes in retinal homogenates. To further characterize the photosensitive nature of cCRY4, we developed an expression system using budding yeast and purified cCRY4 at yields of submilligrams of protein per liter with binding of the flavin adenine dinucleotide (FAD) chromophore. Recombinant cCRY4 dissociated from anti-cCRY4 C1 mAb, which recognizes the C-terminal region of cCRY4, in a light-dependent manner and showed a light-dependent change in its trypsin digestion pattern, suggesting that cCRY4 changes its conformation with light irradiation in the absence of other retinal factors. Combinatorial analyses with UV-visible spectroscopy and immunoprecipitation revealed that there is chromophore reduction in the cCRY4 photocycle and formation of a flavosemiquinone radical intermediate that is likely accompanied by a conformational change in the carboxyl-terminal region. Thus, cCRY4 seems to be an intrinsically photosensitive and photoswitchable molecule and may exemplify a vertebrate model of cryptochrome with possible function as a photosensor and/or magnetoreceptor.
authors.affiliation: Department of Electrical Engineering and Bioscience, Graduate School of Advanced Science and Engineering, Waseda University, Wakamatsu-cho 2-2, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Mitsui
authors.affiliation: Department of Electrical Engineering and Bioscience, Graduate School of Advanced Science and Engineering, Waseda University, Wakamatsu-cho 2-2, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Toshinori
authors.initials: T
authors.lastname: Maeda
authors.affiliation: Department of Electrical Engineering and Bioscience, Graduate School of Advanced Science and Engineering, Waseda University, Wakamatsu-cho 2-2, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Yamaguchi
authors.affiliation: Department of Electrical Engineering and Bioscience, Graduate School of Advanced Science and Engineering, Waseda University, Wakamatsu-cho 2-2, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Tsuji
authors.affiliation: Department of Electrical Engineering and Bioscience, Graduate School of Advanced Science and Engineering, Waseda University, Wakamatsu-cho 2-2, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Ryuji
authors.initials: R
authors.lastname: Watari
authors.affiliation: Department of Electrical Engineering and Bioscience, Graduate School of Advanced Science and Engineering, Waseda University, Wakamatsu-cho 2-2, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Kubo
authors.affiliation: Department of Electrical Engineering and Bioscience, Graduate School of Advanced Science and Engineering, Waseda University, Wakamatsu-cho 2-2, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Okano
authors.affiliation: Department of Electrical Engineering and Bioscience, Graduate School of Advanced Science and Engineering, Waseda University, Wakamatsu-cho 2-2, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Okano
mesh_terms: D000818:Animals; D030161:Avian Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002645:Chickens; D056931:Cryptochromes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008027:Light; D017434:Protein Structure, Tertiary; D012160:Retina / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet
chemical_list: D030161:Avian Proteins; D056931:Cryptochromes; D005182:Flavin-Adenine Dinucleotide