MOLECULAR BASIS FOR C2 DOMAIN-MEMBRANE INTERACTIONS

描述：（逐字来自申请人的摘要） 参与细胞信号传导途径的蛋白质是水溶性的， 只有当它们与膜界面相关联时才发出信号。的 该项目的总体目标是确定结构和部队 控制着蛋白质与膜界面的结合蛋白质结构域 被称为C2结构域的是最普遍的Ca 2+信号转导基序， 真核系统，它们的功能是将蛋白质附着在 膜-溶液界面以Ca 2+依赖的方式。这些领域发挥着 关键作用的过程，如释放脂质衍生的第二个 信使，蛋白磷酸化，膜运输，蛋白质 泛素化、膜孔形成和GT3调节。虽然 这些畴高分辨率溶液和晶体结构是已知的， 关于这些结构域如何在膜上定位并相互作用的信息 与脂质界面的接触是有限的。拟议的工作将决定 来自cPLA 2和突触结合蛋白的C2结构域的方向和位置， 膜界面的定点自旋标记和EPR 谱这种方法也将用于研究结构 伴随Ca 2+和膜结合的变化，以及一种新的方法， 确定静电势将被用来估计电位在 C2结构域的脂质结合表面。这些信息将用于 评估这些结构域的膜附着机制。另一 蛋白质的膜附着机制涉及静电 碱性蛋白基序与酸性脂质界面的相互作用。等 相互作用通常存在于参与细胞信号传导的蛋白质中， 解释了蛋白激酶如src和调节蛋白激酶之间的关系。 蛋白质如MARCKS。拟议的工作将调查的立场， 这些领域内的静电双层和评估的力量 作用于这些将它们连接到膜界面的结构域。我们 预计，更好地了解附着在 蛋白质的膜界面将有助于更好地了解 细胞信号事件的调节和定时。更好地理解 这些相互作用也可能导致开发新的方法， 控制细胞信号传导和细胞生长。

DESCRIPTION: (Verbatim from the Applicant's Abstract) Many proteins that are involved in cell signaling pathways are water-soluble but participate in signaling only when they are associated with the membrane interface. The overall objective of this project is to determine the structures and forces that govern the association of proteins to membrane interfaces. Protein domains known as C2 domains are the most prevalent Ca2+ signaling motif found in eukaryotic systems and they function to attach proteins to the membrane-solution interface in a Ca2+-dependent fashion. These domains play critical roles in processes such as the release of lipid-derived second messengers, protein phosphorylation, membrane trafficking, protein ubiquitination, membrane pore formation, and GTPase regulation. Although high-resolution solution and crystal structures for these domains are known, information on how these domains are positioned on the membrane and interact with the lipid interface is limited. The proposed work will determine the orientation and position of C2 domains from cPLA2 and synaptotagmin on the membrane interface by the use of site-directed spin-labeling and EPR spectroscopy. This methodology will also be used to investigate structural changes that accompany Ca2+ and membrane binding, and a novel approach to determine electrostatic potentials will be used to estimate potentials at the lipid binding surfaces of C2 domains. This information will be utilized to evaluate the mechanisms of membrane attachment of these domains. Another mechanism for the membrane attachment of proteins involves the electrostatic interaction of basic protein motifs with acidic lipid interfaces. Such interactions are commonly found in proteins involved in cell-signaling and they account for the association of protein kinases such as src, and regulatory proteins such as MARCKS. The proposed work will investigate the positions of these domains within the electrostatic double layer and evaluate the forces acting on these domains that attach them to the membrane interface. We anticipate that a better understanding of the forces and mechanisms that attach proteins to the membrane interface will facilitate a better understanding the of the regulation and timing of cell-signaling events. A better understanding of these interactions may also lead to the development of new approaches to control cell-signaling and cell growth.