Transition states in G-protein coupled receptor signaling

描述(申请人提供)：视觉信号转导依赖于GPCR视紫质和异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特征为我们提供了对视觉信号的基本机制的独特见解。然而，对G蛋白信号周期的分子理解还远远不够。我们正在努力揭示信号分子之间形成的复合体的细节。这个目标的一个重要的第一步是确定新的方法来稳定这些正常形成的络合物。我们的第一个目标是稳定视紫红质-G123信号复合体。我们假设该络合物在光激活后将最稳定。这将为未来的生化、结构和体内研究提供一个新的工具，旨在确定信号复合体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这个络合物。目标1：我们将使用交联法稳定视紫红质-转导蛋白复合体。我们已经证明，双(磺基琥珀酰亚胺)琥珀酸双酯(BS3)或其可裂解的类似物3，3‘-二硫双琥珀酰亚胺丙酸酯(DTSSP)可以形成交联键。我们正在努力提高这个反应的效率，并将这个复合体从未反应的视紫红质中提纯出来。交联型视紫红质转导蛋白可用于未来的生化研究，包括电子显微镜或X射线结晶学的结构研究。在目标2中：我们将设计G1I亚基的定点突变体，这些突变体增强了视紫红质的亲和力，并延长了Meta II状态的持续时间。我们已经鉴定了两个具有这些特性的突变体，并将对视紫红质-G1亚单位界面的已知区域进行扫描突变，以确定进一步的突变，以提高这个正常瞬时复合体的稳定性。我们预计，几个突变的组合将稳定视紫红质-G123复合体。使用G1亚单位突变体成功地稳定了该复合体，可用于未来的生化、结构和体内研究。 与公共健康相关：我们正在通过研究G蛋白信号分子之间的瞬时复合体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异三聚体之间的复合体。

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.