Transition states in G-protein coupled receptor signaling

描述（由申请人提供）：视觉信号转导依赖于GPCR视紫红质和异源三聚体鸟嘌呤核苷酸结合蛋白（G蛋白; G1和G23）。视蛋白、视紫红质和G蛋白在多种状态下的结构表征为视觉信号的基本机制提供了独特的见解。然而，对G蛋白信号循环的分子理解还远未完成。我们正在努力揭示信号分子之间形成的复合物的细节。实现这一目标的重要的第一步是确定新的方法来稳定这些通常瞬时形成的复合物。我们的第一个目标是稳定视紫红质- G123信号复合物。我们假设这种复合物在光激活后最稳定。这将为未来的生物化学、结构和体内研究提供新的工具，这些研究旨在确定信号传导复合物的分子结构如何催化核苷酸释放。我们设计了两种互补的方法来稳定该络合物。目的1：我们将使用交联方法来稳定视紫红质-转导素复合物。我们已经表明，可以使用双（磺基琥珀酰亚胺基）辛二酸酯（BS 3）或其可裂解类似物3，3 '-二硫代双琥珀酰亚胺基丙酸酯（DTSSP）形成交联。我们正在努力提高这一反应的效率，并将这种复合物从未反应的视紫红质中纯化出来。交联的视紫红质转导蛋白可用于未来的生化研究，包括通过电子显微镜或X射线晶体学的结构研究。目标2：我们将设计G1 i亚基的定点突变体，这些突变体具有增强的视紫红质亲和力并增加Meta II状态的持续时间。我们已经确定了两个具有这些特性的突变体，并将对视紫红质-G1亚基界面的已知区域进行扫描诱变，以确定进一步的突变，提高这种通常短暂的复合物的稳定性。我们预期几种突变的组合将稳定视紫红质-G123复合物。使用G1亚基突变体的复合物的成功稳定可用于未来的生物化学，结构和体内研究。 公共卫生相关性：我们正致力于通过研究G蛋白信号分子之间的瞬时复合物来确定视觉信号转导的机制。这项为期两年的提案开发了一个模型系统，以稳定G蛋白偶联受体视紫红质和G123异源三聚体之间的复合物。

DESCRIPTION (provided by applicant): Visual signal transduction depends upon the GPCR rhodopsin and heterotrimeric guanine nucleotide binding proteins (G-proteins; G1 and G23). Structural characterization of opsin, rhodopsin, and G-proteins in multiple states has provided exceptional insight into the basic mechanisms of visual signaling. However, the molecular understanding of the G protein signaling cycle is far from complete. We are working to reveal the details of the complexes formed between signaling molecules. An important first step in this goal is to identify new methods to stabilize these normally transiently-formed complexes. Our first target will be stabilization of the rhodopsin- G123 signaling complex. We hypothesize that this complex will be most stable after light activation. This will provide a novel tool for future biochemical, structural and in vivo studies that will aim to identify how the mo- lecular architecture of the signaling complex catalyzes nucleotide release. We have designed two complemen- tary methods for the stabilization of this complex. In Aim 1: We will use cross-linking methods to stabilize the rhodopsin-transducin complex. We have already shown that a cross-link can be formed using bis(sulfosuccinimidyl) suberate (BS3) or its cleavable analog 3,3'- dithiobis-succinimidylpropionate (DTSSP). We are working to improve the efficiency of this reaction and to purify this complex away from unreacted rhodopsin. Cross-linked rhodopsin-transducin can be used for future biochemical studies including structural studies by electron microscopy or x-ray crystallography. In Aim 2: We will design site-directed mutants of the G1i subunit that have enhanced rhodopsin affinity and increase the duration of the meta II state. We have already identified two mutants with these properties, and will perform scanning mutagenesis on known regions of the rhodopsin-G1 subunit interface to identify further mutations that improve the stability of this normally transient complex. We anticipate that combination of several mutations will stabilize the rhodopsin-G123 complex. Successful stabilization of the complex using G1 subunit mutants can be used for future biochemical, structural, and in vivo studies. PUBLIC HEALTH RELEVANCE: We are working to define the mechanisms of visual signal transduction by investigating the transient complexes between G protein signaling molecules. This two year proposal develops a model system to stabilize the complex between the G protein coupled receptor rhodopsin and the G123 heterotrimer.