Structure/Function of Microbial Sensory Rhodopsins

微生物中的视紫红质受体-转导分子复合物已经成为用于研究视紫红质受体-转导分子复合物的范例系统。 了解膜嵌入受体功能在原子分辨率和阐明化学 膜蛋白/蛋白通讯。实验旨在确定之间的协调 我们在当前资助期内在盐古菌中发现的两种分子内信号通路 趋光性受体感觉视紫红质II（SRII）：中间膜的氢键链残基 从光异构化的视黄醛延伸到其膜的第二跨膜螺旋（TM 2）， 嵌入的转换器Htrll，和受体SRII螺旋F和Htrll之间的细胞质相互作用位点。 TM 2的扩展。我们将应用晶体学来定义野生型和信号转导中的结构变化， 变种人时间分辨的光学和振动光谱将被用来映射键的变化， 光活化和通过自旋-自旋偶极耦合测量距离变化的定点自旋标记 对于近残基和双电子-电子共振（DEER）对于远分离的残基。同时，我们 将研究非盐古菌的感觉视紫红质，它表现出非常不同的信号模式比 SR-Htr复合物。鱼腥藻感觉视紫红质（ASR）机制类似于视觉色素/G- 蛋白偶联，因为其转换器是可溶性胞质蛋白（ASRT）。我们得到了晶体结构 的ASR和ASRT在这一时期，我们正在应用诱变和等温滴定量热法， 定义它们的相互作用界面，并进行敲除/拯救实验，以阐明它们的生理功能 in vivo.我们基于ASR光化学的工作假设是，该对调控了 光合作用的触角色素。我们将继续对衣原体的结构/功能进行研究， 感觉视紫红质，其介导Ca++穿过质膜的通量，控制细胞运动。 这些研究的成功将有助于深入了解膜蛋白-蛋白质功能 原子结构和化学机制方面的相互作用，目前在任何膜中都是未知的， 嵌入式分子机器这种相互作用驱动着对正常细胞至关重要的基本膜过程。 细胞功能和膜功能障碍涉及无数疾病状态。新知识将 在原子水平上更好地理解膜分子机制的功能和功能障碍。

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.