Site Directed Spin Labeling of Interfacial Membrane Prot

描述（由申请人提供）：参与细胞信号传导的许多水溶性（界面）蛋白通过与磷脂膜结合在膜表面产生产物来完成。这些蛋白控制着各种细胞反应，如花生四烯酸的释放，用于类二十烷类生物合成，钙信号的第二信使释放，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，并提供了用于膜结合的结构基序的理解。然而，这些研究并没有给出膜结合基序如何与磷脂双分子层相互作用的分子图谱，也没有给出伴随膜结合的结构转变的性质或程度。我们提出的研究使用先进的电子顺磁共振（EPR）光谱，我们开发的新型自旋松弛方法，以及界面蛋白的位点选择性自旋标记，将建立和补充x射线晶体学工作，不仅提供界面蛋白相对于膜的取向，而且提供界面结合基序渗透到膜双层的深度。此外，拟议的研究将定义假设伴随膜结合的结构转变，它们将为未来旨在定义酶催化分子机制的研究奠定基础。目前EPR的研究主要集中在三种与膜结合进行细胞信号传导的重要蛋白：人胞质磷脂酶A2，人磷脂酰肌醇特异性磷脂酶C的d1异构体，以及因子VIII的膜结合部分，该部分是“内在十酶复合物”的一部分。确定蛋白质-膜取向和蛋白质渗透到膜中的深度将提供对特定氨基酸残基促进与特定磷脂组成膜的界面结合作用的分子理解。目前对三种结构表征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插入膜的能量学。这些信息对于理解这些蛋白质在细胞内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靶向特定细胞膜控制的。

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.