Stabilization of Membrane Protein Signaling Complexes

膜蛋白信号复合物的稳定性

• 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
• 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

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）：膜蛋白及其信号传导伴侣之间的复合物结构的确定是一个紧迫的问题，由于缺乏长期稳定复合物的方法而受到阻碍。我们将使用视觉信号转导通路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测复杂的稳定性。视觉信号转导依赖于 GPCR 视紫红质和异三聚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传导的基本机制提供了特殊的见解。然而，由于信号分子之间形成的复合物的细节尚不清楚，因此对 G 蛋白信号循环的分子理解还远未完成。我们的目标是稳定视紫红质-G123 信号复合物和磷酸视紫红质-arrestin1 复合物。这些研究旨在确定哪些因素对于生物瞬时跨膜信号复合物的稳定很重要，这将揭示对于不相关复合物的稳定重要的一般原理。在目标 1 中，我们将鉴定新的 bicelles 混合物组，以提高视紫红质转导蛋白和视紫红质抑制蛋白 1 之间的亲和力。一旦我们确定了可以提高复合物偶联效率和半衰期的 bicelles 混合物，我们将对它们进行结晶试验。在目标 2 中，我们将使用带有负电荷或磷酸化头部基团的标准和新型肽去污剂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。合成后，我们将使用单独的肽去污剂或与胶束去污剂组合来监测每种复合物的稳定性，以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 的修饰对每个复合物亲和力的影响。将对具有改善的亲和力的改变的蛋白质进行结构评估。 公共健康相关性：我们正在努力改进使用视紫红质复合物作为模型系统的瞬态信号复合物的结构研究方法。该提案重点研究模拟生物膜并提高复合物亲和力的膜蛋白溶解试剂。