CONFORMATIONAL ASPECTS OF VISUAL EXCITATION

这个研究计划的总体目标是阐明分子 哺乳动物视网膜视杆细胞视觉兴奋的基础。我们计划 进行下列酶学、光谱和超微结构研究 杆状外节(ROS)的研究：(1)光激活扩增 ROS中的循环涉及光激发的视紫红质、转导蛋白和循环 将对GMP磷酸二酯酶进行详细的研究。其目的是为了 阐明构象转变、动力学和自由能变化 这个周期的每一步。荧光能量转移，纳秒 将进行发射各向异性和红外光散射研究 以绘制亚基结构图并监控亚单位的运动。这个 我们将研究共价修饰的调节作用。(2)我们计划 获得视紫红质配合物的二维晶体阵列 转导蛋白、磷酸二酯酶、视紫红质激酶和其他ROS 蛋白质。这些络合物的分子结构将由 从电子序列重建三维图像 显微照片。(3)霍乱病毒催化转导蛋白的ADP-核糖基化， 阻断其GTP酶活性。将进行肽图绘制以 确定转导蛋白与G蛋白的同源性程度 腺苷环化酶系统。我们想找出它们之间的相似之处 视觉中的信号偶联蛋白和激素作用之间的关系。(4) ROS中的钙调蛋白将被提纯，并与其他 将对蛋白质进行研究，以确定钙敏感基因。这个 钙调素对光激发视紫红质反应能力的影响 转导蛋白催化GTP-GDP交换，对钠钙动力学的影响 膜小泡的交换，以及其催化的反应速度 将测量激酶和磷酸酶。(5)重点工作是 致力于ROS质膜的纯化。离子传输 纯化质膜形成囊泡的性质及其影响 将测量钙离子、钙调蛋白和环状GMP对离子通量的影响。 这些小泡将被用来确定血浆的哪些成分 膜响应于背景照明和瞬变而改变 光脉冲。

The overall goal of this research program is to elucidate the molecular basis of visual excitation in mammalian retinal rod cells. We plan to carry out the following enzymatic, spectroscopic, and ultrastructural studies of rod outer segments (ROS): (1) The light-activated amplification cycle in ROS involving photoexcited rhodopsin, transducin, and the cyclic GMP phosphodiesterase will be investigated in detail. The aim is to elucidate the conformational transitions, kinetics, and free energy change of each step of this cycle. Fluorescence energy transfer, nanosecond emission anisotropy, and infra-red light scattering studies will be carried out to map subunit structure and to monitor the movement of subunits. The regulatory role of covalent modification will be examined. (2) We plan to obtain two-dimensional crystalline arrays of complexes of rhodopsin with transducin, the phosphodiesterase, rhodopsin kinase, and other ROS proteins. The molecular structure of these complexes will be determined by reconstructing three-dimensional images from series of electron micrographs. (3) Cholera catalyzes the ADP-ribosylation of transducin and blocks its GTPase activity. Peptide mapping will be carried out to determine the degree of homology between transducin and the G-protein of the adenylate cyclase system. We want to ascertain the similarities between the signal-coupling proteins in vision and hormone action. (4) Calmodulin from ROS will be purified and its interactions with other proteins will be investigated to identify calcium-sensitive loci. The effects of calcium calmodulin on the capacity of photoexcited rhodopsin to catalyze GTP-GDP exchange in transducin, on the kinetics of sodium-calcium exchange by membrane vesicles, and on the rates of reactions catalyzed by kinases and phosphatases will be measured. (5) A major effort will be devoted to the purification of the ROS plasma membrane. The ion-transport properties of vesicles formed from purified plasma membrane and the effects of calcium ion, calmodulin, and cyclic GMP on ion fluxes will be measured. These vesicles will be used to determine which constituents of the plasma membrane are altered in response to background illumination and transient light pulses.