Membrane Interactions of C2 Domains

蛋白质与细胞内膜界面的可逆结合对于膜运输和细胞信号传导途径的调节至关重要。这种结合激活酶，促进蛋白质-蛋白质相互作用，驱动蛋白质组装的侧向组织，并负责细胞膜的重塑。它也被证明可以调节细胞转化和癌症。膜结合通常由响应于特定细胞信号而与膜界面相关联的专门蛋白质结构域介导;然而，关于由这些结构域进行的膜相互作用的信息很少。拟议的工作将使用EPR为基础的技术称为定点自旋标记，以确定这些域的膜取向和位置。还将评价驱动膜附着的力。突触结合蛋白的C2结构域介导钙依赖性 神经元胞吐作用，以及它们在膜界面上的位置， 将测定磷脂酰肌醇-4，5-二磷酸（一种重要的信号脂质）。静电力可以驱动信号复合物的组装，并且将测试高度带正电荷的蛋白质片段作为在双层表面上螯合蛋白质的支架的假设。两个磷酸肌醇结合结构域的膜位置也将被表征和新的方法来确定蛋白质的取向和膜表面上的深度进行评估。我们预计，更好地了解这些蛋白质-膜相互作用将导致更好地了解膜运输和细胞信号转导事件。对这些事件的更好理解也可能导致控制细胞生长和癌症的新方法的发展。

The reversible binding of proteins to intracellular membrane interfaces is critical to the regulation of membrane trafficking and cell-signaling pathways. This association activates enzymes, facilitates protein-protein interactions, drives the lateral organization of protein assemblies and is responsible for the remodeling of cell membranes. It has also been shown to regulate cell-transformation and cancer. Membrane binding is often mediated by specialized protein domains that associate with the membrane interface in response to specific cellular signals; however, there is little information about the membrane interactions made by these domains. The proposed work will use an EPR based technique termed site-directed spin labeling to determine the membrane orientation and position of these domains. The forces that drive membrane attachment will also be evaluated. The C2 domains of synaptotagmin mediate calcium-dependent neuronal exocytosis, and their positions on the membrane interface in the presence of phosphatidylinositol-4,5-bisphosphate (an important signaling lipid) will be determined. Electrostatic forces may drive the assembly of signaling complexes, and the hypothesis that highly positively charged protein segments act as a scaffold to sequester proteins on the bilayer surface will be tested. The membrane position of two phosphoinositide binding domains will also be characterized and new approaches to determine protein orientation and depth on membrane surfaces evaluated. We anticipate that a better understanding of these protein-membrane interactions will lead to a better understanding of membrane trafficking and cell-signaling events. A better understanding of these events may also lead to the development of new approaches to control cell growth and cancer.