CAREER: Establishing novel signaling transmission modes of LOV photoreceptors

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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.