Division of Material Science, Graduate School of Science , Nagoya University , Chikusa-ku, Furo-cho, Nagoya 464-8602 , Japan.
Department of Earth and Space Science, Graduate School of Science , Osaka University , Osaka 560-0043 , Japan.
J Phys Chem B. 2018 Sep 27;122(38):8819-8823. doi: 10.1021/acs.jpcb.8b05808. Epub 2018 Sep 11.
Formation of the neutral flavin radical in the light-oxygen-voltage-sensing (LOV-sensing) domain of photozipper, based on VfAUREO1, was investigated by electron paramagnetic resonance spectroscopy. The flavin radical was observed in the presence of dithiothreitol by illumination of a LOV-domain mutant (C254S), in which a photoactive cysteine residue in close proximity to flavin was replaced with a serine. The radical did not form under low initial protein-concentration conditions (less than 20 μM). The flavin radicals accumulated with logistic time-dependent kinetics when the protein concentrations were higher than 30 μM. These results indicate that the radical is produced by concerted reactions involving protein interactions and that the radical is formed from the LOV dimer but not the LOV monomer. In contrast, logistic time dependencies were not observed for the sample adapted to the dark following radical formation by illumination, indicating that initialization of the proton pathway is essential for this fast sensing reaction.
基于 VfAUREO1，通过电子顺磁共振波谱法研究了光氧电压感应（LOV-sensing）结构域中光拉链的中性黄素自由基的形成。黄素自由基在二硫苏糖醇存在的情况下通过光照 LOV 结构域突变体（C254S）中观察到，该突变体中靠近黄素的一个光活性半胱氨酸残基被丝氨酸取代。在初始蛋白浓度较低（小于 20 μM）的情况下，自由基不会形成。当蛋白浓度高于 30 μM 时，黄素自由基会以逻辑时间依赖性的方式积累。这些结果表明，自由基是由涉及蛋白质相互作用的协同反应产生的，并且自由基是由 LOV 二聚体形成的，而不是 LOV 单体。相比之下，对于通过光照形成自由基后适应黑暗的样品，没有观察到逻辑时间依赖性，这表明质子途径的初始化对于这种快速感应反应是必要的。
