Stabilization of Membrane Protein Signaling Complexes

描述（由申请人提供）：确定膜蛋白及其信号伙伴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，因为光学读数可以很容易地监测复杂的稳定性。视信号转导依赖于GPCR视紫红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表征为视觉信号传导的基本机制提供了独特的见解。然而，对G蛋白信号周期的分子理解还远未完成，因为信号分子之间形成的复合物的细节尚不清楚。我们的目标将是稳定视紫红质- g123信号复合物和磷酸化视紫红质-阻滞1复合物。这些研究旨在确定哪些因素对生物瞬态跨膜信号复合物的稳定是重要的，这将揭示对不相关复合物稳定重要的一般原理。在目的1中，我们将鉴定出一组新的双胞体混合物，它们可以改善视紫红质转导蛋白和视紫红质抑制蛋白之间的亲和力。一旦我们确定了能提高耦合效率和半衰期的单束混合物，我们将对其进行结晶试验。在目标2中，我们将使用带负电荷或磷酸化头基团的标准和新型肽洗涤剂作为稳定视紫红质-转导蛋白和视紫红质-抑制蛋白1复合物的剂。合成后，我们将使用肽清洗剂单独或与胶束清洗剂联合监测每个复合物的稳定性，以评估其在膜蛋白复合物稳定方面的功效。在目标3中，我们将评估转导蛋白和阻滞蛋白1的修饰对每个复合物亲和力的影响。改善亲和力的改变蛋白将从结构上进行评价。

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.