Stabilization of Membrane Protein Signaling Complexes

膜蛋白信号复合物的稳定性

• 批准号: 8310115
• 负责人: T M Iverson
• 金额: $ 29.7万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8310115
• 关键词: Affinity; Architecture; Arrestins; Binding; Biochemical; Biological; Biological Assay; Biological Models; Cattle; Cells; Charge; Complex; Coupling; Crystallization; Detergents; Electrostatics; Elements; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Proteins; Half-Life; Head; Investigation; Laboratories; Ligand Binding; Lipids; Mediating; Mediator of activation protein; Membrane; Membrane Proteins; Methods; Modification; Molecular; Molecular Conformation; Monitor; Mutation; Opsin; Optics; Pathway interactions; Peptides; Phospholipids; Proteins; Reagent; Receptor Activation; Resolution; Retinal Cone; Rhodopsin; Series; Side; Signal Transduction; Signaling Molecule; Signaling Protein; Site-Directed Mutagenesis; Squid; Structure; Surface; Transducin; Visual; Visual Signal Transduction Pathway; Work; aspartylglutamate; flexibility; functional group; guanine nucleotide binding protein; improved; in vivo; insight; mutant; non-visual arrestins; novel; prevent; protein complex; protein function; retinal rods; stoichiometry

PROJECT SUMMARY Determination of the structures of complexes between membrane proteins and their signaling partners is a pressing problem that has been hindered by the lack of methods for long-term stabilization of the complexes. We will use the visual signal transduction pathway as a model system to develop stabilization strategies since an optical readout can be used to easily monitor complex stability. Visual signal transduction depends upon the GPCR rhodopsin and heterotrimeric guanine nucleotide binding proteins (G-proteins; G¿ and G¿¿). 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 since the details of the complexes formed between signaling molecules are not known. Our targets will be stabilization of the rhodopsin-G¿¿¿ signaling complex and the phosphorhodopsin-arrestin1 com- plex. These studies aim to identify what factors are important for the stabilization of biologically transient transmembrane signaling complexes, which will reveal general principles important for the stabilization of unre- lated complexes. In Aim 1: We will identify novel sets of bicelles mixtures that improve the affinity between rhodopsin-transdicin and rhodopsin-arrestin1. Once we have identified bicelles mixtures that improve the coupling efficiency and half-life of the complex, we will subject these to crystallization trials. In Aim 2: We will use standard and novel peptide detergents with negatively-charged or phosphorylated head groups as agents to stabilize the rhodopsin-transducin and rhodopsin-arrestin1 complexes. Following synthe- sis, we will monitor the stability of each complex using peptide detergents in isolation or in combination with micellar detergents to evaluate their efficacy in stabilization of membrane protein complexes. In Aim 3: We will evaluate the effects of modification of transducin and arrestin1 on the affinity of each com- plex. Altered proteins with improved affinity will be evaluated structurally.

项目概要 确定膜蛋白及其信号伙伴之间的复合物结构是一个 由于缺乏长期稳定复合物的方法，这一紧迫问题受到阻碍。 我们将使用视觉信号转导途径作为模型系统来开发稳定策略 光学读数可用于轻松监测取决于视觉信号转导的复杂稳定性。 GPCR 视紫红质和异三聚鸟嘌呤核苷酸结合蛋白（G 蛋白；G¿ 和 G¿）。 视蛋白、视紫红质和 G 蛋白在多种状态下的表征提供了对视蛋白、视紫红质和 G 蛋白的独特见解。 然而，G 蛋白信号循环的分子理解是。 由于信号分子之间形成的复合物的细节尚不清楚，因此还远未完成。 目标是稳定视紫红质-G¿ ¿ ¿信号复合物和磷酸视紫红质-arrestin1 com- 这些研究旨在确定哪些因素对于生物瞬态的稳定很重要。 跨膜信号复合物，这将揭示对于稳定未重 要的重要的一般原理 迟来的复合物。 目标 1：我们将鉴定新的 bicelles 混合物组，以提高视紫质-转盘素之间的亲和力 一旦我们确定了可以提高偶联效率的 bicelles 混合物， 复合物的半衰期，我们将对它们进行结晶试验。 目标 2：我们将使用带有负电荷或磷酸化头部的标准和新型肽去污剂 基团作为稳定视紫红质转导蛋白和视紫红质抑制蛋白1复合物的试剂。 sis，我们将单独使用肽去垢剂或与 胶束去污剂来评估其稳定膜蛋白复合物的功效。 在目标 3 中：我们将评估转导蛋白和抑制蛋白 1 的修饰对每种成分的亲和力的影响 将对具有改善的亲和力的改变的蛋白质进行结构评估。