SYNTHETIC AND BIOLOGICAL STUDIES OF NUCLEIC ACIDS

重点放在视觉中的光传导上。具体来说，我们将研究 脊椎动物昏暗的光感受器，视紫红质。三个具体的长- 提出了射程目标。 I.信号传递的化学机制(光催化 视网膜异构化)来自七螺旋束(膜结构域) 两个膜外域。分子的构象变化 在视紫红质的照射下，胞质区域出现。这一变化 启动视觉传导的所有生物化学。我们的目标是 理解这种变化的确切化学本质。 II.信号放大的机制。关于结合力的研究 GDP-转导后视紫红质II，GTP交换GDP和释放 塔尔法GTP。这将包括以下联系人的详细映射 视紫红质和不同形式的转导蛋白。 生物化学和光适应机制。理解 适应是主要的最终目标。以下是整体流程 导致视觉传导的脱敏和终止不是 理解：1、单磷酸化和/或单磷酸化位点的选择 视紫红质激酶(RK)对视紫红质的多聚磷酸化作用；2，机制 磷酸化视紫红质与arrestin的相互作用 其他未知蛋白质；3，这些蛋白质/蛋白质是如何相互作用的 抢占以下所需的结合部位和结构 信号放大(变紫红质II-转导蛋白相互作用)。 我们能理解伴随而来的蛋白质构象变化吗 参与这些进程？ 四、实验方法。这些将是跨学科的，跨越 有机化学、生物化学、分子生物学和生物物理学(本刊 与生物物理研究小组的合作)。方法包括 人工合成视紫红质基因的广泛突变、表达和 表达的突变体蛋白的纯化、化学研究 突变体，包括分子间和分子内交联，使用 光敏和其他双官能团。介绍一到两个 特定部位的半胱氨酸残基及其作为化学品处理的用途 和生物物理(荧光，EPR)研究。关于考古遗址的研究 RK、转导蛋白和arrestin与变视紫质的结合部位。 磷酸化位点的操控及其局限性 视紫红质由广泛的氨基酸替代，作用单一V/S Arrestin结合中的多个磷酸化作用。这些研究将包括 转导蛋白、RK和arrestin与变视紫质结合的开关速率 II及其磷酸化形式。

The focus is on light transduction in vision. Specifically, we will study the dim light vertebrate photoreceptor, rhodopsin. Three specific long- range goals are proposed. I. Chemical Mechanism of the Transmission of the Signal (Light-catalyzed Retinal Isomerization) from the Seven Helical Bundle (the Membrane Domain) to the Two Extra-membranous Domains. A conformational change in the cytoplasmic domain occurs on illumination of rhodopsin. This change initiates all the biochemistry of the visual transduction. Our goal is to understand the precise chemical nature of this change. II. Mechanisms in Amplification of the Signal. Study of the binding of GDP-transducin to Metarhodopsin II, exchange of GDP by GTP and release of Talpha GTP. This will include detailed mapping of the contacts between rhodopsin and different forms of transducin. III. Biochemistry and Mechanisms of Adaptation to Light. Understanding adaptation is the major ultimate goal. The following overall processes leading to desensitization and termination of visual transduction are not understood: 1, selection of sites of single phosphorylation and/or polyphosphorylations of rhodopsin by rhodopsin kinase (RK); 2, mechanisms of interactions of phosphorylated rhodopsin with arrestin and possibly other proteins as yet unknown; 3, how do these protein/protein interactions pre-empt the binding sites and structures that are necessary for amplification of the signal (Metarhodopsin II- transducin interaction). Can we understand the accompanying conformational changes in the proteins participating in these processes? IV. Experimental Approaches. These would be interdisciplinary, spanning organic chemistry, biochemistry, molecular biology and biophysics (this in collaborations with Biophysical research groups). Approaches involve extensive mutagenesis in the synthetic rhodopsin gene, expression and purification of the expressed mutant proteins, chemical studies of the mutants including inter and intra-molecular crosslinking, using photosensitive and other bifunctional groups. Introduction of one or two cysteine residues at specific sites and their use as handles for chemical and biophysical (fluorescence, EPR) studies. Studies of the sites of the binding sites of RK, transducin and arrestin to Metarhodopsin. Manipulation as well as limitations of the phosphorylation sites in rhodopsin by extensive amino acid replacements, the role of single V/S multiple phosphorylations in arrestin binding. These studies will include on and off rates of binding of transducin, RK and arrestin to Metarhodopsin II and its phosphorylated forms.