EPR and Solid-State NMR Studies of Integral Membrane Proteins

完整膜蛋白的 EPR 和固态 NMR 研究

基本信息

批准号：
8135980
负责人：
GARY A LORIGAN
金额：
$ 25.75万
依托单位：
MIAMI UNIVERSITY OXFORD
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-28 至 2013-08-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Membrane proteins are responsible for many important properties and functions of biological systems: they transport ions and molecules across the membrane, they act as receptors, and they have roles in the assembly, fusion, and structure of cells and viruses. Despite the abundance and clear importance of membrane-associated molecules, very little structural information about these systems exists. The objective of the proposed research is to develop new EPR structural biology methods to probe the structural and dynamic properties of integral membrane proteins. The novel experiments proposed in this study will reveal new insights concerning the structural characteristics of phospholamban (PLB) in lipid bilayers and develop new biophysical methods utilizing bicelles. The NIH has recognized the importance of studying the structural properties of integral membrane proteins with PA-06-119. This program announcement has specifically requested for new biophysical techniques to probe the structures of membrane proteins. In accordance with this program announcement, new EPR spectroscopic methods will be developed to probe the structures of integral membranes and directly compare our results with solid-state NMR spectroscopic data. This proposal is method development in nature; thus, the integral membrane protein PLB will be used a model membrane protein system. We feel that this new approach will move the field forward so that researchers can more easily and inexpensively determine the structural topology of integral membrane proteins using spin-label EPR spectroscopy. The specific aims of this proposal consist of the following: (1) Develop a new spin- label EPR spectroscopy technique to determine the helical tilt of integral membrane peptides inside bicelles using PLB as a model; (2) Develop alternative membrane protein alignment methods using mechanically oriented phospholipid bilayers and phospholipid bilayer nanotube arrays using spin-label EPR spectroscopy; (3) Determine the orientation and secondary structure of phospholamban with respect to the phospholipid bilayer using solid-state NMR spectroscopy; and (4) Investigate the membrane-bound protein dynamics of phospholamban with spin-label EPR spectroscopy and solid-state NMR spectroscopy.Project Narrative The NIH has recognized the importance of studying the structural properties of integral membrane proteins with PA-06-119. This program announcement has specifically requested for new biophysical techniques to probe the structures of membrane proteins. In accordance with this program announcement, we will develop new EPR spectroscopic methods to probe the structures of integral membranes and directly compare our results with solid-state NMR spectroscopy to validate this new approach. This proposal is method development in nature; thus, PLB will be used as a model membrane protein system.

描述（由申请人提供）：膜蛋白负责生物系统的许多重要特性和功能：它们跨膜运输离子和分子，它们充当受体，并且它们在细胞和细胞的组装、融合和结构中发挥作用。病毒。尽管膜相关分子丰富且重要性明显，但有关这些系统的结构信息却很少。本研究的目的是开发新的 EPR 结构生物学方法来探测整合膜蛋白的结构和动态特性。本研究中提出的新颖实验将揭示有关脂质双层中受磷蛋白（PLB）结构特征的新见解，并利用 bicelles 开发新的生物物理方法。 NIH 已经认识到使用 PA-06-119 研究整合膜蛋白结构特性的重要性。该计划公告特别要求采用新的生物物理技术来探测膜蛋白的结构。根据该计划公告，将开发新的 EPR 光谱方法来探测整体膜的结构，并将我们的结果与固态 NMR 光谱数据直接进行比较。该提案本质上是方法开发；因此，整合膜蛋白PLB将被用作模型膜蛋白系统。我们认为这种新方法将推动该领域向前发展，以便研究人员能够使用自旋标记 EPR 光谱更轻松、更经济地确定整合膜蛋白的结构拓扑。该提案的具体目标包括以下内容：（1）开发一种新的自旋标记 EPR 光谱技术，以 PLB 作为模型确定 bicelles 内完整膜肽的螺旋倾斜； (2) 利用机械定向磷脂双层和利用自旋标记 EPR 光谱的磷脂双层纳米管阵列开发替代膜蛋白排列方法； (3) 使用固态核磁共振波谱确定受磷蛋白相对于磷脂双层的取向和二级结构； (4) 利用自旋标记 EPR 光谱和固态 NMR 光谱研究受磷蛋白的膜结合蛋白动力学。项目叙述 NIH 已经认识到使用 PA-06-119 研究整合膜蛋白结构特性的重要性。该计划公告特别要求采用新的生物物理技术来探测膜蛋白的结构。根据该计划公告，我们将开发新的 EPR 波谱方法来探测整体膜的结构，并将我们的结果与固态核磁共振波谱直接进行比较，以验证这种新方法。该提案本质上是方法开发；因此，PLB将被用作模型膜蛋白系统。