Stabilization of Membrane Protein Signaling Complexes

膜蛋白信号复合物的稳定性

基本信息

批准号：
8028067
负责人：
T M Iverson
金额：
$ 30万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
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/8028067
关键词：
Affinity Architecture Arrestins Binding Biochemical Biological Biological Assay Biological Models Cattle Cells Charge Complex Coupled Coupling Crystallization Detergents Electrostatics Elements 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 Protein Subunits 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

项目摘要

DESCRIPTION (provided by applicant): 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; 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 since the details of the complexes formed between signaling molecules are not known. Our targets will be stabilization of the rhodopsin-G123 signaling complex and the phosphorhodopsin-arrestin1 complex. 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 unrelated complexes. In Aim 1 we will identify novel sets of bicelles mixtures that improve the affinity between rhodopsin-transducin 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 synthesis, 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. PUBLIC HEALTH RELEVANCE: We are working to improve the methods for structural investigation of the transient signaling complexes using complexes of rhodopsin as a model system. This proposal focuses on investigating solubilization reagents for membrane proteins that mimic biological membranes and improve complex affinity.

描述（由申请人提供）：确定膜蛋白质及其信号伴侣之间复合物的结构是一个紧迫的问题，由于缺乏长期稳定复合物的方法而阻碍了膜的问题。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，因为光学读数可用于轻松监视复杂的稳定性。视觉信号转导取决于GPCR视紫红质和异三聚素鸟嘌呤核苷酸结合蛋白（G蛋白； G1和G23）。多种状态下，Opsin，Rhodopsin和G蛋白的结构表征为视觉信号传导的基本机制提供了出色的见解。但是，由于尚不清楚信号传导分子之间形成的复合物的细节，因此对G蛋白信号周期的分子理解远非完整。我们的目标将是稳定Rhodopsin-G123信号传导复合物和磷光蛋白 - arrestin1复合物。这些研究旨在确定哪些因素对于稳定生物瞬时跨膜信号传导复合物很重要，这将揭示对于稳定不相关复合物很重要的一般原理。在AIM 1中，我们将确定一组新型的双层混合物，这些混合物可以改善Rhodopsin-Transducin和Rhodopsin-arrestin1之间的亲和力。一旦我们确定了提高复合物的耦合效率和半衰期的双层混合物，我们将进行结晶试验。在AIM 2中，我们将使用带负荷或磷酸化的头部基团的标准和新型肽洗涤剂作为稳定Rhodopsin-Transducin和Rhodopsin-artrestin1配合物的药物。合成后，我们将使用肽洗涤剂分离或与胶束洗涤剂结合使用肽洗涤剂来监测每个复合物的稳定性，以评估其在膜蛋白复合物稳定中的疗效。在AIM 3中，我们将评估转杜蛋白和抑制素的修饰对每个复合物的亲和力的影响。将在结构上评估具有改善亲和力的蛋白质的改变。公共卫生相关性：我们正在努力改善使用视紫红蛋白作为模型系统的复合物对瞬态信号传导复合物进行结构研究的方法。该建议着重于研究模仿生物膜并改善复杂亲和力的膜蛋白的溶解试剂。