The mechanism of signal transduction in photoreceptors

描述（由申请人提供）：信号接收和信号转导构成活细胞感知并最终对外部刺激（如光、化学物质（营养素、毒素、激素）、热和机械信号）作出反应的基本过程。大多数信号受体存在于细胞质膜中，细胞质膜将细胞内部与外部介质分开。光的初级感受器（光感受器）是所有视觉形式的基础，其中来自吸收的单光子的能量用于引起发色团的构象变化，通常是皮秒内双键的异构化。在较慢的时间尺度上，周围的蛋白质和水基质响应于发色团的构象变化，并最终导致由换能器分子感测的构象和静电变化。微生物视紫红质是一类光活性、七跨膜螺旋、亚视黄基蛋白质，存在于嗜盐古菌、变形菌、蓝藻、真菌和藻类等多种微生物中。盐古菌中的感觉视紫红质I和II（SRI和SRII）、衣原体视紫红质CsoA和CsoB以及鱼腥藻视紫红质是感光受体，在整个可见光谱中进行光谱调谐以将关于环境中光的强度和颜色的信息传递给细胞。SRI和SRII是盐生嗜盐杆菌及其相关嗜盐古菌的趋光性感受器。SRI介导的引诱剂运动响应的绿色橙色波长所使用的离子泵BR和HR，而SRII介导的蓝光回避反应。SRI和SRII蛋白是多组分信号传导复合物的亚基，并通过蛋白质-蛋白质相互作用将信号传递到整合的膜转导蛋白（分别为HtrI和HtrII），所述膜转导蛋白控制调节细胞运动装置的细胞质磷酸化途径。为了阐明光感受器感觉视紫红质（SRII）的光谱调谐的基础，并获得一个详细的结构为基础的机制，光诱导的构象变化如何触发信号在其同源换能器（HtrII），我们将1。确定单位点突变体R72 A的结构，这是一种最近与光谱调谐有关的残基。2、确定光感受器在长寿命（O）信号状态下的晶体结构，并通过将其与最近解决的基态结构进行比较来识别主要信号组分。3、比较9-TM螺旋SRII/HtrII融合构建体在关闭状态和光激活信号状态下的晶体结构，为了确定最近鉴定的鱼腥藻光感受器及其可溶性推定的转换器的原子结构和光激活机制，我们将4.确定可溶性推定转换器的晶体结构，这是一种与感光器相同操纵子上的14 kDa蛋白质，与任何已知蛋白质都没有序列同源性。5.确定初级鱼腥藻光感受器在基态和信号状态下的晶体结构。6.确定鱼腥藻光感受器与其推定的换能器的复合物的结构。

DESCRIPTION (provided by applicant): Signal reception and signal transduction constitute fundamental processes by which living cells perceive and ultimately react to external stimuli such as light, chemicals (nutrients, toxins, hormones), heat and mechanical signals. Most signal receptors reside in the cytoplasmic membrane that separates the inside of a cell from the external medium. Primary receptors for light (photoreceptors) are the basis of all forms of vision where the energy from absorbed single photons is used to bring about a conformational change in the chromophore, usually the isomerization of a double bond within picoseconds. On a slower timescale, the surrounding protein and water matrix responds to the conformational change of the chromophore and ultimately results in conformational and electrostatic changes that are sensed by a transducer molecule. Microbial rhodopsins are a family of photoactive, seven-transmembrane helix, retinylidene proteins found in phylogenetically diverse microorganisms, including haloarchaea, proteobacteria, cyanobacteria, fungi, and algae. Sensory rhodopsins I and II (SRI and SRII) in haloarchaea, Chlamydomonas rhodopsins CsoA and CsoB, and Anabaena rhodopsin, are photosensory receptors, spectrally tuned throughout the visible spectrum to relay information to the cell regarding the intensity and color of light in the environment. SRI and SRII are sensors for phototaxis in Hatobacterium salinarum and related halophilic archaea. SRI mediates attractant motility responses to green-orange wavelengths used by the ion pumps BR and HR, while SRII mediates blue-light avoidance responses. The SRI and SRII proteins are subunits of multicomponent signaling complexes and relay signals by protein-protein interaction to integral membrane transducer proteins (HtrI and HtrII, respectively) that control a cytoplasmic phosphorylation pathway that modulates the cell's motility apparatus. In order to elucidate the basis of spectral tuning of the photoreceptor sensory rhodopsin (SRII) and to obtain a detailed structure-based mechanism of how light-induced conformational changes trigger signaling in its cognate transducer (HtrII) we will 1. Determine the structure of the single-site mutant R72A, a residue recently implicated in spectral tuning. 2, Determine the crystal structure of the photoreceptor in the long-lived (O) signaling state and identify the main signaling components by comparing it to the recently solved ground state structure. 3, Compare the crystal structure of the 9-TM helical SRII/HtrlI fusion construct in the off-state with that of the photoactivated signaling state, In order to determine the atomic structure and the photoactivation mechanism of the recently identified Anabaena photoreceptor and its soluble putative transducer we will 4. Determine the crystal structure of the soluble putative transducer, a 14-kDa protein on the same operon as the photoreceptor with no sequence homology to any known protein. 5. Determine the crystal structure of the primary Anabaena photoreceptor in the ground and signaling state. 6. Determine the structure of the complex of the Anabaena photoreceptor with its putative transducer.