Membrane Interactions of C2 Domains

蛋白质与细胞内膜界面的可逆结合对膜运输和细胞信号通路的调控至关重要。这种结合激活酶，促进蛋白质之间的相互作用，推动蛋白质组装的横向组织，并负责细胞膜的重塑。它还被证明可以调节细胞转化和癌症。膜结合通常由与膜界面相关的特定蛋白质结构域来调节，以响应特定的细胞信号；然而，关于这些结构域所产生的膜相互作用的信息很少。这项拟议的工作将使用一种基于EPR的技术，称为位置定向自旋标记来确定这些结构域的膜方向和位置。还将评估驱动膜附着的力。突触素的C2结构域介导钙依赖 神经细胞胞吐作用及其在膜界面上的位置 将测定磷脂酰肌醇-4，5-二磷酸(一种重要的信号脂质)。静电力可能会驱动信号复合体的组装，高正电荷蛋白质片段作为支架将蛋白质隔离在双层表面的假设将得到检验。还将表征两个磷脂酰肌醇结合结构域的膜位置，并评价确定膜表面蛋白质方向和深度的新方法。我们预计，对这些蛋白质-膜相互作用的更好理解将导致对膜运输和细胞信号事件的更好理解。更好地了解这些事件也可能导致开发新的方法来控制细胞生长和癌症。

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.