SYNTHETIC AND BIOLOGICAL STUDIES OF NUCLEIC ACIDS

重点是视觉中的光传导。 具体来说，我们将研究 脊椎动物的弱光光感受器，视紫红质。 三个具体的长- 提出了范围目标。 I. 光催化信号传递的化学机理 视网膜异构化）从七螺旋束（膜域） 两个膜外领域。 的构象变化 胞质结构域在视紫红质的照射下出现。 这种变化 启动视觉传导的所有生物化学过程。 我们的目标是 了解这种变化的确切化学性质。 二. 信号放大的机制。 的结合研究 GDP-转导至后视紫红质II，通过GTP交换GDP并释放 塔阿尔法GTP。 这将包括详细绘制 视紫红质和不同形式的转导素。 三. 光适应的生物化学和机制。 理解 适应是主要的最终目标。 以下整体流程 导致视觉传导的脱敏和终止， 理解：1，选择单一磷酸化位点和/或 视紫红质激酶（RK）对视紫红质的多磷酸化作用; 2，机制 磷酸化视紫红质与抑制蛋白的相互作用， 其他蛋白质还未知; 3，这些蛋白质/蛋白质相互作用如何 抢占结合位点和结构是必要的， 信号的放大（后视紫红质II-转导素相互作用）。 我们能理解伴随蛋白质的构象变化吗 参与这些进程？ 四. 实验方法。 这些将是跨学科的， 有机化学，生物化学，分子生物学和生物物理学（这在 与生物物理研究小组合作）。 方法涉及 在合成视紫红质基因、表达和 表达的突变蛋白的纯化， 突变体，包括分子间和分子内交联，使用 光敏和其它双官能团。 介绍一两个 在特定位点的半胱氨酸残基及其作为化学试剂的手柄的用途 和生物物理（荧光，EPR）研究。 研究的网站， RK、转导蛋白和抑制蛋白与后视紫红质的结合位点。 磷酸化位点的操作和限制 视紫红质经广泛氨基酸置换，作用为单V/S 抑制蛋白结合中的多重磷酸化。 这些研究将包括 transformin、RK和arrestin与后视紫红质结合的开启和关闭速率 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.