Site Directed Spin Labeling of Interfacial Membrane Prot

描述(申请人提供)：一些参与细胞信号传递的水溶性(界面)蛋白通过与磷脂膜结合在膜表面产生产物来实现这一点。这些蛋白质控制细胞的各种反应，如花生四烯酸的生物合成，第二信使的释放，钙信号的释放，以及细胞表面触发的血液凝固。界面蛋白的高分辨率X射线结构的数量正在增加，并提供了对用于膜结合的结构基序的理解。然而，这些研究没有给出膜结合基序如何与磷脂双层相互作用的分子图像，也没有给出伴随膜结合的结构转变的性质或程度。我们提出的使用先进的电子顺磁共振(EPR)光谱、我们开发的新的自旋弛豫方法以及界面蛋白的位置选择性自旋标记的研究将建立在X射线结晶学工作的基础上，并通过提供界面蛋白相对于膜的取向以及界面结合基序穿透到膜双层的深度来补充X-射线结晶学工作。此外，拟议的研究将定义假设伴随着膜结合的结构转变，并将为未来旨在确定酶催化的分子机制的研究奠定基础。目前的EPR研究集中在三个与细胞信号转导的膜结合的重要蛋白质上：人胞浆磷脂酶A2，人磷脂酰肌醇特异性磷脂酶C的D_1亚型，以及因子的膜结合部分，它是“内在十酶复合体”的一部分。确定蛋白质-膜的取向和蛋白质穿透膜的深度将有助于从分子上了解特定氨基酸残基在促进特定磷脂组成的膜的界面结合方面的作用。目前对三种结构良好的酶的研究将为该项目的长期目标提供重要的见解，该目标是了解驱动界面酶膜插入的能量学。这一信息对于理解这些蛋白质在细胞中或细胞表面的作用很重要，因为这些酶的功能部分是由它们定位于特定的细胞膜控制的。

DESCRIPTION (provided by applicant): A number of water-soluble (interfacial) proteins involved in cell signaling do so by binding to phospholipid membranes to produce products at the membrane surface. Such proteins control various cellular responses, such as the release of arachidonic acid for biosynthesis of eicosanoids, second messenger liberation for calcium signaling, and cell-surface triggered blood coagulation. The number of high-resolution X-ray structures of interfacial proteins is increasing, and is providing an understanding of the structural motifs used for membrane binding. However, such studies do not give a molecular picture of how membrane binding motifs interact with phospholipid bilayers nor the nature or extent of structural transitions that accompany membrane binding. Our proposed studies using advanced electron paramagnetic resonance (EPR) spectroscopy, novel spin relaxant methods that we have developed, and site-selective spin-labeling of interfacial proteins will build on and complement the X-ray crystallographic work by providing not only the orientation of the interfacial proteins with respect to the membrane but also the depth of penetration of the interfacial binding motifs into the membrane bilayer. In addition, the proposed studies will define structural transitions that are hypothesized to accompany membrane binding and they will set the stage for future studies aimed at defining the molecular mechanisms of enzyme catalysis. The current EPR studies focus on three important proteins that bind to the membrane for cell signaling: human cytosolic phospholipase A2, the d1 isoform of human phosphatidylinositol-specific phospholipase C, and the membrane-binding portion of factor VIII, which is part of the "intrinsic ten-ase complex". Determining the protein-membrane orientation and depth of protein penetration into the membrane will provide a molecular understanding of the roles of specific amino acid residues to promote interfacial binding to membranes of specific phospholipid composition. The current studies on three structurally well-characterized enzymes will provide important insights related to a long-term goal for this project which is to understand the energetics that drive membrane insertion of interfacial enzymes. This information is important in understanding the action of these proteins in cells or on cell surfaces because the function of these enzymes is controlled in part by their targeting to specific cell membranes.