Stabilization of Membrane Protein Signaling Complexes

描述（由申请人提供）：膜蛋白及其信号传导伴侣之间复合物结构的测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化策略，因为光学读数可以用于容易地监测复杂的稳定性。视觉信号转导依赖于GPCR视紫红质和异源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的基本机制提供了独特的见解。然而，对G蛋白信号循环的分子理解远未完成，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信号复合物和磷视紫红质-arrestin 1复合物。这些研究旨在确定哪些因素对生物瞬时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的稳定很重要的一般原则。在目标1中，我们将确定新的双胞混合物，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bicelles混合物，提高耦合效率和复合物的半衰期，我们将这些结晶试验。在目标2中，我们将使用标准的和新的肽洗涤剂与负电荷或磷酸化的头部基团作为代理，以稳定视紫红质-transducin和视紫红质-arrestin 1复合物。合成后，我们将使用肽洗涤剂单独或与胶束洗涤剂组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化中的功效。在目标3中，我们将评估转导素和抑制素1的修饰对每个复合物的亲和力的影响。将在结构上评价具有改善的亲和力的改变的蛋白。 公共卫生相关性：我们正在努力改进的方法，用于结构调查的瞬态信号复合物使用复合物的视紫红质作为一个模型系统。该提案的重点是研究模拟生物膜和提高复合物亲和力的膜蛋白的增溶试剂。

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.