CAREER: Establishing novel signaling transmission modes of LOV photoreceptors

光感受器是一种复杂的蛋白质机器，它对一种基本的感觉输入做出反应：光。它们几乎存在于所有生物体中，对行为、昼夜节律、新陈代谢和光合作用至关重要。因此，阐明它们的功能和工作方式(I)告知从细菌到人类的有机体如何适应环境变化和维持生命，以及(Ii)为设计“光遗传”蛋白质工具提供设计规则，这些工具用于用光控制细胞功能。该职业奖的总体目标是通过光生物学、合成生物学和生物工程的统一研究和教育计划，揭示光/氧/电压(LOV)光感受器信号在自然和工程系统中的新机制和应用。该项目将以G蛋白信号调节因子(RGS)为本体论效应器，建立LOV蛋白的信号功能和信号传递模式。RGS-LOV的存在一直是基于生物信息学的假设，但从未被生化和生物物理表征所证实。初步数据表明，光驱动的结构重排揭示了一个膜定位序列，它增强了RGS与其G-α信号伙伴之间的蛋白质-蛋白质相互作用，从而在易位时终止G-蛋白偶联受体信号。RGS-LOV的结构-功能关系和功能信号活性将被研究：(I)使用从细菌中产生的纯化的重组蛋白进行体外研究；(Ii)通过光谱分析、免疫组织化学和高分辨率显微镜对异源表达的哺乳动物细胞进行细胞分析。在天然LOV蛋白中，既没有观察到光诱导膜定位，也没有观察到G蛋白调节子与光门控膜定位在同一蛋白中的功能分离。这项研究将通过传播光遗传工具和开源硬件来影响广泛的研究界。教育活动将通过注重实践学习和将湿实验室经验与生物信号数学模型相结合的活动，将LOV光生物学的研究和应用扩展到本科生研究和STEM培训。

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.