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的复合体的结构是一个紧迫的问题，由于缺乏长期稳定复合体的方法而受到阻碍。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。视觉信号转导依赖于GPCR视紫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特征为我们提供了对视觉信号的基本机制的独特见解。然而，由于信号分子之间形成的复合体的细节尚不清楚，因此对G蛋白信号周期的分子理解还远远不完整。我们的目标将是稳定视紫红质-G123信号复合体和磷酸视紫质-抑制素1复合体。这些研究旨在确定哪些因素对生物瞬时跨膜信号复合体的稳定起重要作用，这将揭示对无关复合体稳定重要的一般原理。在目标1中，我们将确定能够提高视紫红质转导蛋白和视紫红质抑制蛋白1之间亲和力的新型双环混合物。一旦我们确定了可以提高偶联效率和络合物半衰期的双电极混合物，我们将对这些混合物进行结晶试验。在目标2中，我们将使用带有负电荷或磷酸化头基的标准和新型多肽洗涤剂作为试剂来稳定视紫红质转导蛋白和视紫红质抑制蛋白1复合体。合成后，我们将单独使用多肽洗涤剂或与胶束洗涤剂结合使用来监测每个复合体的稳定性，以评估它们稳定膜蛋白复合体的效果。在目标3中，我们将评估转导蛋白和阻止蛋白1的修饰对每个复合体亲和力的影响。亲和力提高的改变的蛋白质将从结构上进行评估。 公共卫生相关性：我们正在努力改进使用视紫红质复合体作为模型系统的瞬时信号复合体的结构研究方法。这项建议的重点是研究膜蛋白的增溶试剂，以模拟生物膜并提高络合亲和力。

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.