Rhodopsin receptor-transducer molecular complexes in microorganisms have become paradigm systems for understanding membrane-embedded receptor function at atomic resolution and elucidating the chemistry of membrane protein/protein communication. Experiments are designed to identify the coordination between the two intramolecular signaling pathways we identified in the current funding period in the haloarchaeal phototaxis receptor sensory rhodopsin II (SRII): the mid-membrane hydrogen-bonded chain of residues extending from the photoisomerizing retinal to the second transmembrane helix (TM2) of its membrane- embedded transducer Htrll, and the cytoplasmic interaction site between receptor SRII helix F and the extension of TM2. We will apply crystallography to define structural changes in wild-type and signaling mutants. Time-resolved optical and vibrational spectroscopy will be used to map bond alterations during photoactivation, and site-directed spin-labeling to measure distance changes by spin-spin dipolar coupling for near residues and double electron-electron resonance (DEER) for far separated residues. In parallel we will study the non-haloarchaeal sensory rhodopsins, which exhibit very different modes of signaling than the SR-Htr complexes. The Anabaena sensory rhodopsin (ASR) mechanism is analogous to visual pigment/G- protein coupling in that its transducer is a soluble cytoplasmic protein (ASRT). We obtained crystal structures of ASR and ASRT in this period, and we are applying mutagenesis and isothermal titration calorimetry to define their interaction interface, and knock out/rescue experiments to elucidate their physiological function in vivo. Our working hypothesis based on ASR photochemistry is that the pair regulates biosynthesis of the antenna pigments of photosynthesis. We will continue structure/function studies of the Chlamydomonas sensory rhodopsins, which mediate Ca++ fluxes across the plasma membrane controlling cell motility. Success of these studies promises a depth of understanding of membrane protein-protein functional interactions in terms of atomic structure and chemical mechanism currently unknown in any membrane- embedded molecular machine. Such interactions drive fundamental membrane processes crucial to normal cellular function, and membrane dysfunction is involved in myriad disease states. The new knowledge will better our understanding of function and dysfunction of membrane molecular machinery at the atomic level.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (NSS)
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Chin, Jean
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University of Texas Health Science Center Houston
Schools of Medicine
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Sineshchekov, Oleg A; Govorunova, Elena G; Wang, Jihong et al. (2013) Intramolecular proton transfer in channelrhodopsins. Biophys J 104:807-17
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