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Diverse Roles of Glycine Residues Conserved in Photoactive Yellow Proteins

Yasushi Imamoto ^* , Sanae Tatsumi , Miki Harigai  , Yoichi Yamazaki , Hironari Kamikubo  and Mikio Kataoka 

^* Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan; Graduate School of Materials Science, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan; and Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University, Inuyama, Aichi 484-8506, Japan

Correspondence: Address reprint requests to Yasushi Imamoto, Dept. of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan. Tel.: 81-75-753-4243; Fax: 81-75-753-4210; E-mail: imamoto{at}vision-kyoto-u.jp.

The role of glycine residues was studied by alanine-scanning mutagenesis using photoactive yellow protein, a structural prototype of PER ARNT SIM domain proteins, as a template. Mutation of glycine located close to the end of β-strands with dihedral angles disallowed for alanine (Gly-37, Gly-59, Gly-86, and Gly-115) induces destabilization of the protein structure. On the other hand, substitution for Gly-77 and Gly-82, incorporated into the fifth -helix, slows the photocycle by 15–20 times, suggesting that these residues regulate the light-induced structural switch between dark-state structure and signaling-state structure. Most importantly, a significant amount of G29A is in the bleached state and showed a 1000-fold slower photocycle. As O₂ of the carboxylic acid of Glu-46 is close enough for contact with C of Gly-29, alanine mutation perturbs this packing. Fourier transform infrared spectroscopy demonstrated that the C=O₂ stretching mode of Glu-46 is 6 cm^–1 upshifted in G29A, suggesting that C of Gly-29 acts as a proton donor for the C-H···O₂ hydrogen bond with Glu-46, which stabilizes the dark-state structure. During the photocycle, Glu-46 becomes negatively charged by donating a proton to the chromophore, resulting in breakage of this hydrophobic packing and consequent conformational change of the protein.