CAREER: Establishing novel signaling transmission modes of LOV photoreceptors

光感受器是复杂的蛋白质机器，对基本的感觉输入：光做出反应。它们存在于几乎所有生物体中，对行为、昼夜节律、新陈代谢和光合作用至关重要。因此，阐明它们做什么以及它们如何工作（i）告知从细菌到人类的生物体如何适应环境变化并维持生命，以及（ii）为工程“光遗传学”蛋白质工具提供设计规则，以控制光的细胞功能。这个CAREER奖的总体目标是揭示光/氧/电压（LOV）感光细胞信号传导在自然和工程系统中的新机制和应用，通过光生物学，合成生物学，本项目将建立LOV蛋白的信号传导功能和信号传导模式，以及G蛋白信号传导调节因子（RGS）作为它们的本体效应器。RGS-LOV的存在是基于生物信息学假设的，但从未被生物化学和生物物理表征证实。初步数据表明，光驱动的结构重排揭示了膜定位序列，其增强了RGS与其G-α信号传导伴侣之间的蛋白质-蛋白质相互作用，以在易位时终止G-蛋白偶联受体信号传导。将（i）使用细菌中产生的纯化重组蛋白在体外研究RGS-LOV结构-功能关系和功能信号活性，以及（ii）使用异源表达哺乳动物细胞中的细胞测定，通过光谱学、免疫组织化学和高分辨率进行评估显微镜。在天然LOV蛋白中，既没有观察到光诱导的膜定位，也没有观察到G-蛋白和光门控膜定位在一个蛋白中的调节子之间的功能分离。该研究将通过光遗传学工具和开源硬件的传播影响广泛的研究社区。教育活动将LOV光生物学研究和应用扩展到本科研究和STEM培训，通过注重实践学习的活动，并将湿实验室经验与生物信号的数学模型相结合。

Photoreceptors are complex protein machines that respond to a fundamental sensory input: light. They are found in nearly all organisms and are critical to behavior, circadian rhythms, metabolism, and photosynthesis. Thus, elucidating what they do and how they work (i) informs how organisms from bacteria to humans adapt to environmental changes and sustain life, and (ii) provides design rules for engineering "optogenetic" protein tools for controlling cellular function with light. The overall goal of this CAREER Award is to reveal novel mechanisms and applications of light/oxygen/voltage (LOV) photoreceptor signaling in natural and engineered systems, through a unified research and educational program at the interface of photobiology, synthetic biology, and bioengineering.This project will establish the signaling function and signal transmission mode of LOV proteins with regulators of G-protein signaling (RGS) as their ontological effectors. The existence of RGS-LOV has been hypothesized based on bioinformatics but never proven by biochemical and biophysical characterization. Preliminary data suggests that the light-driven structural rearrangements unmask a membrane localization sequence, which enhances protein-protein interaction between the RGS and its G-alpha signaling partner to terminate G-protein coupled receptor signaling upon translocation. RGS-LOV structure-function relationships and functional signaling activity will be studied (i) in vitro using purified recombinant protein produced in bacteria and (ii) using cellular assays in heterologously expressing mammalian cells, as assessed by spectroscopy, immunohistochemistry, and high-resolution microscopy. Neither light-induced membrane localization nor the functional separation between regulators of G-proteins and light-gated membrane localization in one protein has been observed among natural LOV proteins. The research will impact broad research communities through the dissemination of optogenetic tools and open-source hardware. Educational activities will extend LOV photobiology studies and application into undergraduate research and STEM training, through activities that focus on hands-on learning and combining wet laboratory experience with mathematical models of biological signaling.