EPR and Solid-State NMR Studies of Integral Membrane Proteins

完整膜蛋白的 EPR 和固态 NMR 研究

• 批准号: 7925719
• 负责人: GARY A LORIGAN
• 金额: $ 26.01万
• 依托单位: MIAMI UNIVERSITY OXFORD
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-28 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7925719
• 关键词: Binding Proteins; Cellular Structures; Characteristics; Collaborations; Data; Electron Spin Resonance Spectroscopy; Glass; Integral Membrane Protein; Ion Transport; Label; Lipid Bilayers; Membrane; Membrane Proteins; Methods; Modeling; NIH Program Announcements; NMR Spectroscopy; Nature; Pattern; Peptides; Phospholipids; Property; Protein Dynamics; Proteins; Research; Research Personnel; Role; Sampling; Side; Signal Transduction; Site; Spin Labels; Structure; Structure-Activity Relationship; System; Techniques; United States National Institutes of Health; Vertebral column; Virus; base; biological systems; insight; magnetic field; method development; novel; novel strategies; phospholamban; protein protein interaction; receptor; research study; solid state nuclear magnetic resonance; structural biology; two-dimensional

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：它们在整个膜上运输离子和分子，它们充当受体，并且在细胞和结构的组装，融合和结构中具有作用病毒。尽管与膜相关的分子具有丰富和清晰的重要性，但有关这些系统的结构信息很少。 拟议研究的目的是开发新的EPR结构生物学方法来探测整体膜蛋白的结构和动态特性。这项研究中提出的新实验将揭示有关脂质双层中磷脂（PLB）结构特征的新见解，并利用双皮细胞开发了新的生物物理方法。 NIH认识到研究具有PA-06-119的整体膜蛋白的结构特性的重要性。该程序公告专门要求采用新的生物物理技术来探测膜蛋白的结构。根据此计划公告，将开发新的EPR光谱方法来探测积分膜的结构，并将我们的结果与固态NMR NMR光谱数据直接进行比较。该提议本质上是方法的发展。因此，整体膜蛋白PLB将用于模型膜蛋白系统。我们认为这种新方法将向前进，以便研究人员可以使用自旋标签EPR光谱法更轻松，更便宜地确定整体膜蛋白的结构拓扑。 该提案的具体目的包括以下内容：（1）开发一种新的自旋标记EPR光谱技术，以使用PLB作为模型来确定Bicelles内部膜肽的螺旋倾斜度； （2）使用机械定向的磷脂双层和磷脂双层纳米管阵列开发替代膜蛋白比对方法； （3）使用固态NMR光谱法确定相对于磷脂双层相对于磷脂双层的方向和二级结构； （4）研究具有自旋标签的EPR光谱和固态NMR光谱的磷脂的膜结合蛋白动力学。项目叙事 NIH认识到研究具有PA-06-119的整体膜蛋白的结构特性的重要性。该程序公告专门要求采用新的生物物理技术来探测膜蛋白的结构。根据此计划公告，我们将开发新的EPR光谱方法来探测积分膜的结构，并将我们的结果与固态NMR光谱进行直接比较，以验证这种新方法。该提议本质上是方法的发展。因此，PLB将用作模型膜蛋白系统。