MECHANISM OF VISUAL EXCITATION

描述（改编自申请人的摘要）：长期目标 本项目旨在阐明生物化学和生物物理机制 潜在的视觉兴奋和感光细胞代谢。的方法 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开始于 视色素和测距到活体视网膜的NMR研究。具体 目的包括（1）视色素的结构：（A）建模 视觉色素的结构（a）绘制氨基酸衬 通过将发色团共价连接到 特异性半胱氨酸;（B）表征二价阳离子结合位点 视紫红质，调查锥状色素是否有这样的网站， 确定阳离子结合是否具有任何生理后果;（c） 开发一个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 使用氨基放置的半经验观察进行建模 膜蛋白中的酸。(B)一个圆锥体的特征- 型色素，P521，通过（a）获得振动光谱 正常状态和贫Cl状态下色素的发色团; (b)确定糖部分的序列和组成 （c）定位P521的Cl-结合位点， （d）表征P521的磷酸化， 体内光吸收(2)控制席夫碱的pKa， 其反离子：（A）阐明水在确定 （B）测定Schiff碱中的关键氨基酸， 控制席夫碱的pK的视黄酸结合位点;（C） 确定质子化的反离子的效果， 脊椎动物和无脊椎动物的视觉色素，并确定阴离子 在某些情况下可以作为反离子。(3)光化学 （A）确定章鱼的抗衡离子是否 视紫红质在光吸收后变成质子化，并将 质子化动力学变化;（B）确定氘化是否影响 漂白中间体之间的转变动力学，和 阐明所发生的质子化变化;以及（C）阐明 氯离子对锥型颜料光漂白的影响（四） 含锥体感光膜的静电特性 类型颜料及其后果：（A）测量符号和 含有壁虎P521的表面电荷密度的大小 光感受器膜，并确定膜是否为阳性 带电荷的（B）表征与壁虎P521相关的转导蛋白; 将其测序;将其序列中与 激活的P521;和（5）视网膜的体内NMR研究：（A）31 P-NMR （a）观察体内转导素上的GDP/GTP交换;（B） 观察cGMP在体内的变化及光、钙对cGMP的调节作用; (B)质子NMR研究（a）获得蟾蜍的水抑制光谱 视网膜并鉴定主峰，尤其是乳酸盐;（B）观察 并量化光照对5a中所确定物种的影响;以及 (c)观察并量化氧气、葡萄糖和钙的影响 在5A中识别的物种。

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.