MECHANISM OF VISUAL EXCITATION

描述(改编自申请者摘要)：长期目标 本项目旨在阐明生物化学和生物物理机制。 潜在的视觉兴奋和光感受器代谢。该方法 是在几个层面上解决重要的相关问题 光感受器细胞的组织始于 视网膜的视觉色素和核磁共振研究。特定的 目标包括(1)视觉色素的结构：(A)模拟 通过(A)绘制出氨基酸衬里的视觉色素的结构 通过将生色团共价连接到 特定半胱氨酸；(B)二价阳离子结合部位的特征 视紫红质，调查视锥色素是否有这样的位置，以及 确定阳离子结合是否有任何生理后果；(C) 为可视化开发详细的三维结构模型 基于实验约束和计算机相结合的颜料 用半经验观察法对氨基的放置进行建模 膜蛋白中的酸。(B)刻画锥体的性质- 壁虎P521的典型色素，通过(A)获得振动光谱 正常和缺氯状态下的色素的生色团； (B)确定碳水化合物部分的顺序和组成 该色素；(C)定位P521的氯离子结合部位。 发色团；(D)表征P521的磷酸化 体内的光吸收。(2)席夫碱的pKA控制和 它的反离子：(A)阐明水在决定 席夫碱的pK；(B)测定 控制席夫碱PK的视网膜结合部位；(C) 测定两者反离子的质子化效应 脊椎动物和无脊椎动物的视觉色素，并确定阴离子 在某些情况下可以起到反离子的作用。(3)光化学 视觉色素：(A)测定章鱼的反离子 视紫红质在光吸收后质子化，并分解 质子化发生动力学变化；(B)确定重氢是否影响 漂白中间体之间过渡的动力学，以及 澄清发生的质子化变化；以及(C)阐明 氯离子对锥形颜料光漂白的影响。(4) 含锥体光感受器膜的静电特性 类型颜料及其后果：(A)测量标志和 含有壁虎P521的表面电荷密度的大小 光感受器膜，并确定膜是否呈阳性 带电(B)鉴定与壁虎P521相关的转导蛋白； 测序；比较其序列中与之相互作用的部分 活化的P521；和(5)视网膜的活体核磁共振研究：(A)31P-核磁共振 研究(A)观察体内转导蛋白上的GDP/GTP交换； 体内观察cGMP，观察光和钙对其调节作用； (B)质子核磁共振研究(A)获得蟾蜍的水抑制谱 视网膜，并确定主要峰值，特别是乳酸；(B)观察 并量化光照对5a中确定的物种的影响；以及 (C)观察和量化氧气、葡萄糖和钙的影响 关于5a中确定的物种。

DESCRIPTION (Adapted from applicant's abstract): The long term goal of this project is to elucidate the biochemical and biophysical mechanisms underlying visual excitation and photoreceptor metabolism. The approach is to attack important, related problems at several levels of organization of the photoreceptor cell starting at the level of the visual pigment and ranging to NMR studies of retina in vivo. Specific aims include (1) the structure of visual pigments: (A) Modeling the structure of visual pigments by (a) mapping out the amino acids lining the chromophore binding site by covalently linking the chromophore to specific cysteines; (b) characterizing the divalent cation binding site of rhodopsin, investigating if cone pigments have such a site, and determining if cation binding has any physiological consequences; (c) developing a detailed three dimensional structural model for visual pigments based on a combination of experimental constraints and computer modeling using semi-empirical observations on the placement of amino acids in membrane proteins. (B) Characterizing the properties of a cone- type pigment from gecko, P521 by (a) obtaining the vibrational spectrum of the chromophore of the pigment in its normal and Cl-depleted states; (b) determining the sequence and composition of carbohydrate moiety of the pigment; (c) localizing the Cl- binding site of P521 with respect to the chromophore; (d) characterizing the phosphorylation of P521 after light absorption in vivo. (2) Control of the pKa of the Schiff base and its Counter ion: (A) elucidating the role of water in determining the pK of the Schiff base; (B) determining the key amino acids in the retinal binding site which control the pK of the Schiff base; (C) determining the effect of protonation of the counter ion of both vertebrate and invertebrate visual pigments, and determining if anions can serve as a counter ion under some circumstances. (3) Photochemistry of Visual Pigments: (A) determining if the counter ion of octopus rhodopsin becomes protonated after light absorption, and resolving the protonation changes kinetically; (B) determining if deuteration affects the kinetics of the transitions between the bleaching intermediates, and clarifying the protonation changes that occur; and (C) elucidating the effect of chloride ions on the photobleaching of cone- type pigments. (4) Electrostatic Properties of Photoreceptor Membranes Containing a Cone Type Pigment and their Consequences: (A) measuring the sign and magnitude of the surface charge density of gecko P521-containing photoreceptor membranes, and determining if the membrane is positively charged (B) characterizing the transducin associated with gecko P521; sequencing it; comparing the part of its sequence which interacts with activated P521; and (5) in vivo NMR Studies of the Retina: (A) 31P-NMR studies to (a) observe GDP/GTP exchange on transducin in vivo; (b) observe cGMP in vivo and observe its modulation by light and calcium; (B) Proton NMR studies (a) to obtain water suppressed spectra of toad retinas and identify the major peaks, especially lactate; (b) to observe and quantify the effects of light on the species identified in 5a; and (c) to observe and quantify the effects of oxygen, glucose and calcium on the species identified in 5a.