MOLECULAR BASIS FOR C2 DOMAIN-MEMBRANE INTERACTIONS

C2 域-膜相互作用的分子基础

• 批准号: 6228434
• 负责人: DAVID S CAFISO
• 金额: $ 20万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6228434
• 关键词: binding sites; biological signal transduction; calcium ion; cell cell interaction; cell membrane; chemical bond; conformation; cysteine; electron spin resonance spectroscopy; ionic bond; membrane activity; membrane lipids; membrane structure; molecular site; mutant; phospholipase A2; protein binding; protein structure function; site directed mutagenesis; structural biology; surface property; synaptotagmin

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.

描述：(逐字摘自申请者的摘要)许多蛋白质是 参与细胞信号通路是水溶性的，但参与 只有当它们与膜界面相关联时才会发出信号。这个 本项目的总体目标是确定结构和受力 它控制着蛋白质与膜界面的结合。蛋白质结构域 被称为C2结构域的是在 真核系统，它们的功能是将蛋白质连接到 膜-溶液界面以钙离子依赖的方式。这些领域发挥作用 在脂类第二次释放等过程中的关键作用 信使，蛋白质磷酸化，膜转运，蛋白质 泛素化、膜孔形成和GTP酶调节。虽然 这些区域的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和相互作用的信息 与脂质的界面是有限的。拟议的工作将决定 CPLA2和突触素中C2结构域的取向和位置 用定点自旋标记和EPR研究膜界面 光谱学。这种方法也将被用来研究结构 伴随着钙离子和膜结合的变化，以及一种新的方法 确定静电势将用于估计 C2结构域的脂质结合面。这些信息将被用来 评估这些结构域的膜附着机制。另一个 蛋白质的膜附着机制与静电有关 碱性蛋白质模体与酸性脂类界面的相互作用。是这样的 相互作用通常存在于参与细胞信号传递的蛋白质中，它们 解释蛋白激酶的关联，如src，和调节 蛋白质，如Marcks。拟议的工作将调查 这些区域内的静电双电层，并评估作用力 作用于这些结构域，将它们连接到膜界面。我们 预计更好地了解与之相关的力量和机制 蛋白质到膜的界面将有助于更好地理解 细胞信号事件的调节和时间安排。更好地理解 这些互动还可能导致开发新的方法来 控制细胞信号和细胞生长。