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：
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
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/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

项目摘要

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.

项目摘要确定膜蛋白及其信号伴侣之间复合物结构的确定是缺乏长期稳定复合物的方法阻碍的紧迫问题。我们将使用视觉信号传输途径作为模型系统来制定稳定策略，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视紫红质和异三元鸟嘌呤核肽结合蛋白（G蛋白； g蛋白； g蛋白和g。）。结构在多种状态下，Opsin，Rhodopsin和G蛋白的特征表征为对该的特殊见解视觉信号的基本机制。但是，对G蛋白信号周期的分子理解是远非完整，因为尚不清楚信号分子之间形成的复合物的细节。我们的目标将是稳定Rhopopostin-g ??信号传导复合物和磷光蛋白 - arrestin1 com- plex。这些研究旨在确定哪些因素对于稳定生物瞬时很重要跨膜信号传导复合物，该复合物将揭示对于稳定未重复的一般原则层压复合物。在AIM 1中：我们将确定一组新型的双层混合物，以改善Rhodopsin-Transdicin之间的亲和力和Rhodopsin-arrestin1。一旦我们确定了提高耦合效率和综合体的半衰期，我们将进行结晶试验。在AIM 2中：我们将使用否定的或磷酸化的头部使用标准和新型肽决定作为稳定视紫红质 - 透明蛋白和视紫红蛋白 - arrestin1配合物的剂量的组。遵循综合 SIS，我们将使用肽隔离或与胶束决定评估其在膜蛋白复合物稳定方面的效率。在AIM 3中：我们将评估跨多霉素和抑制素的修饰对每个com的亲和力的影响 plex。将在结构上评估具有改善亲和力的蛋白质的改变。