Stabilization of Membrane Protein Signaling Complexes

项目总结 膜蛋白与其信号伙伴之间的复合体结构的测定是一种 这是一个紧迫的问题，由于缺乏长期稳定这些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制定稳定策略，因为 光学读数可用于轻松监控复杂的稳定性。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；G？和G？？)。结构性 视蛋白、视紫红质和G蛋白在多种状态下的特征为我们提供了对 视觉信号的基本机制。然而，对G蛋白信号周期的分子理解是 由于信号分子之间形成的复合体的细节尚不清楚，因此这一研究还远未完成。我们的 目标将是稳定视紫红质-G？信号复合体和磷酸视紫红质-阻滞素-1复合体。 普莱克斯。这些研究的目的是确定哪些因素对生物瞬变的稳定是重要的 跨膜信号转导复合体的研究将揭示对细胞外信号转导的稳定具有重要意义的一般规律。 迟来的复合体。 在目标1中：我们将确定一组新的双细胞混合物，以提高视紫红质-转运蛋白之间的亲和力 和视紫红质抑制素1。一旦我们确定了提高偶联效率和提高偶联效率的双通道混合物 半衰期，我们将对这些进行结晶试验。 目标2：我们将使用带负电荷或磷酸化头部的标准和新型多肽洗涤剂 作为稳定视紫红质转导蛋白和视紫红质抑制蛋白1复合体的试剂。在Synthe之后- SIS，我们将单独或结合使用多肽洗涤剂来监测每个复合体的稳定性 胶束洗涤剂用于评价其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COM亲和力的影响。 普莱克斯。亲和力提高的改变的蛋白质将从结构上进行评估。

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.