Mechanism of signal transduction in photoreceptors

描述(申请人提供)：信号接收和信号转导是活细胞感知外部刺激并最终对其做出反应的基本过程，如光、化学物质(营养物质、毒素、激素)、热量和机械信号。大多数信号受体驻留在将细胞内部与外部介质分开的细胞质膜上。初级光感受器(光感受器)是所有形式视觉的基础，其中来自被吸收的单光子的能量被用来引起发色团的构象变化，通常是在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对发色团的构象变化做出反应，最终导致构象和静电变化，由换能器分子感知。微生物视紫红蛋白是一类具有光活性的七跨膜螺旋视黄醇蛋白，存在于不同的微生物系统发育中，包括嗜盐古细菌、蛋白细菌、蓝藻、真菌和藻类。在海洋古生菌中，感觉视紫红质I和II(SRI和SRII)，衣藻视紫红质CsoA和CSOB，以及鱼腥藻视紫红质，是光感受器，在整个可见光谱中进行光谱调节，向细胞传递关于环境中光的强度和颜色的信息。SRI和SRII是盐藻及其相关嗜盐古细菌趋光性的感受器。SRI介导对离子泵BR和HR所使用的绿橙色波长的吸引运动反应，而SRII介导蓝光回避反应。SRI和SRII蛋白是多组分信号复合体的亚基，通过蛋白质-蛋白质相互作用将信号传递到完整的膜转导蛋白(分别为HtrI和HtrII)，控制细胞质磷酸化途径，调节细胞的运动装置。为了阐明光感受器感觉视紫红质(SRII)的光谱调谐的基础，并获得光诱导的构象变化如何触发其同源转导(HtrII)中的信号的详细的基于结构的机制，我们将1.确定单位点突变体R72a的结构，这是最近与光谱调谐有关的残基。2、确定长寿命(O)信号态的光感受器的晶体结构，并通过将其与最近求解的基态结构进行比较来识别主要的信号成分。3、比较9-TM螺旋SRII/HtrlI融合结构在关闭状态和光激活信号状态下的晶体结构，以确定最近发现的鱼腥藻光受体及其可溶性转导蛋白的原子结构和光激活机制。4.确定可溶性转导蛋白的晶体结构，该转导蛋白位于与光感受器相同的操纵子上，与任何已知蛋白质没有序列同源性。5.测定鱼腥藻初级感光体在基态和信号态的晶体结构。6.确定鱼腥藻感光蛋白复合体的结构及其可能的转导。

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.