EPR and Solid-State NMR Studies of Integral Membrane Proteins

描述(申请人提供)：膜蛋白负责生物系统的许多重要性质和功能：它们跨膜运输离子和分子，它们作为受体，它们在细胞和病毒的组装、融合和结构中发挥作用。尽管膜相关分子数量丰富且具有明显的重要性，但关于这些系统的结构信息却很少。 这项研究的目的是开发新的EPR结构生物学方法来探索完整的膜蛋白的结构和动力学性质。这项研究中提出的新颖实验将揭示磷蛋白(PLB)在脂质双层中的结构特征，并开发利用双分子的新的生物物理方法。美国国家卫生研究院已经认识到用PA-06-119研究完整膜蛋白结构性质的重要性。这项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。根据这一计划公告，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体核磁共振波谱数据进行直接比较。这一建议本质上是方法的发展，因此，完整的膜蛋白PLB将被用来作为膜蛋白系统的模型。我们认为，这种新的方法将推动这一领域的发展，使研究人员能够更容易和廉价地使用自旋标记EPR光谱来确定完整膜蛋白的结构拓扑。 这项建议的具体目标包括：(1)开发一种新的自旋标记EPR波谱技术，以PLB为模型来确定整体膜多肽在双分子链中的螺旋倾斜度；(2)利用自旋标记EPR光谱技术，开发使用机械定向磷脂双层和磷脂双层纳米管阵列的替代膜蛋白排列方法；(3)利用固体核磁共振光谱确定磷蛋白相对于磷脂双层的取向和二级结构；以及(4)利用自旋标记EPR光谱和固态核磁共振光谱研究磷蛋白与膜结合的蛋白质动力学。 美国国家卫生研究院已经认识到用PA-06-119研究完整膜蛋白结构性质的重要性。这项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。根据这一计划公告，我们将开发新的EPR波谱方法来探测整体膜的结构，并直接将我们的结果与固态核磁共振波谱进行比较，以验证这一新方法。这一建议本质上是方法的发展，因此，PLB将被用作膜蛋白系统的模型。

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.