EPR and Solid-State NMR Studies of Integral Membrane Proteins

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：它们跨膜转运离子和分子，它们作为受体，并且它们在细胞和病毒的组装、融合和结构中发挥作用。尽管膜相关分子的丰度和明确的重要性，很少有关于这些系统的结构信息存在。 该研究的目的是开发新的EPR结构生物学方法来探测膜蛋白的结构和动力学特性。在这项研究中提出的新的实验将揭示新的见解受磷蛋白（PLB）在脂质双层的结构特征，并开发新的生物物理方法利用bicelles。NIH已经认识到用PA-06-119研究整合膜蛋白的结构特性的重要性。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光谱方法来探测完整膜的结构，并将我们的结果与固态NMR光谱数据直接进行比较。该提议本质上是方法开发;因此，将使用整合膜蛋白PLB作为模型膜蛋白系统。我们认为，这种新的方法将推动该领域的发展，使研究人员可以更容易，更便宜地确定使用自旋标记EPR光谱完整的膜蛋白的结构拓扑结构。 该提案的具体目标包括以下内容：（1）开发新的自旋标记EPR光谱技术以使用PLB作为模型来确定双胞内的完整膜肽的螺旋倾斜;（2）使用自旋标记EPR光谱技术开发使用机械取向的磷脂双层和磷脂双层纳米管阵列的替代膜蛋白质对齐方法;（3）使用固态NMR光谱确定受磷蛋白相对于磷脂双层的取向和二级结构;（4）使用自旋标记EPR光谱和固态NMR光谱研究受磷蛋白的膜结合蛋白动力学。 NIH已经认识到用PA-06-119研究整合膜蛋白的结构特性的重要性。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光谱方法来探测完整膜的结构，并将我们的结果与固态NMR光谱直接比较，以验证这种新方法。该提议本质上是方法开发;因此，PLB将被用作模型膜蛋白系统。

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.