Site Directed Spin Labeling of Interfacial Membrane Prot

界面膜保护的定点旋转标记

• 批准号: 6943630
• 负责人: Bruce H Robinson
• 金额: $ 23.42万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6943630
• 关键词: X ray crystallography; active sites; binding proteins; binding sites; biological signal transduction; electron spin resonance spectroscopy; enzymes; lipid bilayer membrane; membrane proteins; molecular site; phosphatidylinositols; phospholipase A2; phospholipase C; protein binding; protein structure function

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.

描述（由申请人提供）：通过与磷脂膜结合以在膜表面生产产物，参与细胞信号传导的许多水溶性（界面）蛋白是这样做的。这种蛋白质控制了各种细胞反应，例如甘氨酰酸释放用于类eicosanoids的生物合成，用于钙信号传导的第二信使释放以及细胞表面触发的血液凝结。界面蛋白的高分辨率X射线结构的数量正在增加，并且正在提供对用于膜结合的结构基序的理解。但是，此类研究并未给出分子图，即膜结合基序如何与磷脂双层相互作用，也没有伴有膜结合的结构过渡的性质或程度。我们提出的研究使用先进的电子顺磁共振（EPR）光谱法，我们开发的新型自旋松弛方法以及对界面蛋白质的现场选择性自旋标记将基于X射线晶体学工作，并补充X射线晶体学工作，不仅通过提供对膜片的跨性别蛋白的方向，还可以通过提供对膜片的跨性别构件的方向，并提供对束缚的构图。双层。此外，拟议的研究将定义结构过渡，这些跃迁伴随膜结合，它们将为未来的研究奠定阶段，以定义酶催化的分子机制。当前的EPR研究集中于三种与膜结合的细胞信号传导结合的重要蛋白：人类胞质磷脂酶A2，人磷脂酰肌醇特异性磷脂酶C的D1同工型，以及VIII因子VIII的膜结合部分，这是“本质中的Ten-Ten-asase复合物”的一部分。确定蛋白质膜的方向和蛋白质渗透到膜中的深度将提供对特定氨基酸残基的作用的分子理解，以促进界面与特定磷脂组成的膜结合。当前对三种结构良好的酶的研究将提供与该项目的长期目标相关的重要见解，该酶是了解驱动界面酶插入膜插入的能量学。该信息对于理解这些蛋白在细胞或细胞表面的作用很重要，因为这些酶的功能部分通过其靶向特定细胞膜来控制。