Fluorescent proteins based on light, oxygen, and voltage (LOV) sensing photoreceptors are among the few reporter gene technologies available for studying living systems in oxygen‐free environments that render reporters based on the green fluorescent protein nonfluorescent. LOV reporters develop fluorescence by binding flavin mononucleotide (FMN), which they endogenously obtain from cells. As FMN is essential to cell physiology as well as for determining fluorescence in LOV proteins, it is important to be able to study and characterize flavin binding in LOV reporters. To this end, we report a method for reversibly separating FMN from two commonly used LOV reporters to prepare stable and soluble apoproteins. Using fluorescence titration, we measured the equilibrium dissociation constant for binding with all three cellular flavins: FMN, flavin adenine dinucleotide, and riboflavin. Finally, we exploit the riboflavin affinity of apo LOV reporters, identified in this work, to develop a fluorescence turn‐on biosensor for vitamin B2.

中文翻译：

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不依赖氧的荧光报告基因中黄素结合的表征

基于光、氧和电压 (LOV) 传感光感受器的荧光蛋白是少数可用于研究无氧环境中生命系统的报告基因技术之一，该技术使基于绿色荧光蛋白的报告基因不发出荧光。LOV 报告基因通过结合从细胞内源获得的黄素单核苷酸 (FMN) 来发出荧光。由于 FMN 对于细胞生理学以及确定 LOV 蛋白中的荧光至关重要，因此能够研究和表征 LOV 报告基因中的黄素结合非常重要。为此，我们报告了一种从两种常用的 LOV 报告基因中可逆分离 FMN 的方法，以制备稳定且可溶的脱辅基蛋白。使用荧光滴定，我们测量了与所有三种细胞黄素结合的平衡解离常数：FMN、黄素腺嘌呤二核苷酸和核黄素。最后，我们利用本工作中确定的 apo LOV 报告基因的核黄素亲和力来开发维生素 B2 的荧光开启生物传感器。