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：（逐字研究申请人的摘要）许多蛋白质 参与细胞信号通路是水溶性的，但参与 仅当它们与膜界面相关联时。这 该项目的总体目的是确定结构和力量 控制蛋白质与膜界面的关联。蛋白质结构域 称为C2域是在 真核系统，它们起作用可将蛋白固定在 以Ca2+依赖性方式进行膜溶液界面。这些域播放 在过程中的关键作用，例如脂质衍生的第二 信使，蛋白质磷酸化，膜运输，蛋白质 泛素化，膜孔的形成和GTPase调节。虽然 这些结构域的高分辨率溶液和晶体结构已知， 有关这些域如何在膜上定位并相互作用的信息 使用脂质接口有限。拟议的工作将确定 CPLA2和突触符号的C2域的方向和位置 膜界面通过使用位置定向的自旋标记和EPR 光谱法。该方法还将用于研究结构 伴随Ca2+和膜结合的变化，以及一种新颖的方法 确定静电电位将用于估计电位 C2结构域的脂质结合表面。此信息将用于 评估这些结构域的膜附着的机制。其他 蛋白质膜附着的机理涉及静电 碱性蛋白基序与酸性脂质界面的相互作用。这样的 相互作用通常在参与细胞信号的蛋白质中发现，它们是 解释蛋白质激酶（例如SRC）和调节的关联 蛋白质，例如马克克斯。拟议的工作将调查 静电双层内的这些域并评估力 作用于将它们连接到膜接口的这些域。我们 预计对附加力和机制有更好的理解 膜接口的蛋白质将有助于更好地理解 细胞信号事件的调节和时机。更好的理解 这些相互作用也可能导致新方法的发展 控制细胞信号和细胞生长。