Interplay of Light, Redox Potential and Temperature in Light-Oxygen-Voltage Receptors

感觉光感受器赋予生物体对光的敏感性，它们在光遗传学中兼作基因可编码致动器，通过光对细胞生理学进行精确控制。光氧电压（LOV）光感受器通过黄素核苷酸吸收蓝光，触发典型的光循环，这需要在严格保守的半胱氨酸残基和黄素发色团之间形成硫醚键。缺乏这种半胱氨酸，LOV受体表现出增强的荧光和活性氧（ROS）的产生；出乎意料的是，缺乏半胱氨酸的LOV受体能够通过蓝光诱导形成黄素中性半醌自由基状态进行下游信号转导。在此背景下，我们将阐明在含半胱氨酸和不含半胱氨酸的LOV受体中各种输入（光、温度和氧化还原电位）和输出（荧光、ROS产生、信号转导）的相互作用。光谱电化学、生化和结构分析将确定控制这些过程的分子决定因素，从而允许合理构建具有优化信号响应和最小化副反应性的增强受体。通过将对光以外信号的敏感性嫁接到LOV受体上，可以设计出新颖的、精确控制的细胞电路。天然的蛋白质库包括多个与LOV受体具有显著同源性的入口，但缺乏一个或几个通常保守的残基。对这些假设受体的深入表征将进一步加深我们对自然界中LOV受体日益多面的作用的理解，并为增强对光、氧化还原电位和/或温度敏感的衍生受体的工程设计提供蓝图。

Sensory photoreceptors endow organisms with sensitivity to light, and they double as genetically encodable actuators in optogenetics for the precise control by light of cellular physiology. Light-oxygen-voltage (LOV) photoreceptors absorb blue light by flavin nucleotides to trigger a canonical photocycle which entails formation of a thioether bond between a strictly conserved cysteine residue and the flavin chromophore. Lacking this cysteine, LOV receptors exhibit enhanced fluorescence and generation of reactive oxygen species (ROS); unexpectedly, cysteine-devoid LOV receptors are capable of downstream signal transduction via blue-light-induced formation of a flavin neutral sem-iquinone radical state. Against this backdrop, we will elucidate the interplay of various inputs (light, temperature and redox potential) and outputs (fluorescence, ROS production, signal transduction) in both cysteine-containing and cysteine-free LOV receptors. Spectroelectrochemical, biochemical and structural analyses will identify molecular determinants governing these processes, in turn allowing the rational construction of enhanced receptors with optimized signal response and minimized side reactivity. By grafting sensitivity to signals other than light onto LOV receptors, novel, precisely con-trollable cellular circuits can be devised. The natural repertoire of proteins comprises multiple entries with significant homology to LOV receptors but lacking one or several normally conserved residues. Mechanistic insight stemming from an in-depth characterization of these putative receptors will fur-ther our understanding of the increasingly multi-facetted roles of LOV receptors in Nature and stand to provide blueprints for the engineering of enhanced derivative receptors sensitive to light, redox potential and/or temperature.