Stabilization of Membrane Protein Signaling Complexes

项目摘要 确定膜蛋白及其信号传导伙伴之间的复合物的结构是一个重要的研究方向。 这是一个紧迫的问题，由于缺乏长期稳定络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， 可以使用光学读出器来容易地监测复合稳定性。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; G）。结构 视蛋白、视紫红质和G蛋白在多种状态下的表征提供了对 视觉信号的基本机制。然而，对G蛋白信号循环的分子理解是 这远未完成，因为信号分子之间形成的复合物的细节还不清楚。我们 目标将是稳定视紫红质-G蛋白信号复合物和磷酸视紫红质-抑制蛋白1复合物， 丛这些研究旨在确定哪些因素对生物学短暂性的稳定是重要的。 跨膜信号复合物，这将揭示一般原则的重要稳定unre- 复杂的。 在目标1中：我们将确定新的双胞混合物，提高视紫红质-transdicin之间的亲和力， 和视紫红质抑制蛋白1。一旦我们确定了提高耦合效率的双胞混合物， 配合物的半衰期，我们将对这些进行结晶试验。 在目标2中：我们将使用带有负电荷或磷酸化头部的标准和新型肽洗涤剂 基团作为稳定视紫红质-转导素和视紫红质-抑制素1复合物的试剂。在合成之后- SIS，我们将使用肽去污剂单独或与 胶束去污剂以评价它们在稳定膜蛋白复合物中的功效。 在目标3中：我们将评估转导素和抑制素1的修饰对每种化合物亲和力的影响。 丛将在结构上评价具有改善的亲和力的改变的蛋白。

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.