Structural studies of membrane proteins by NMR

描述（由申请人提供）：本提案的最终目标是为通过溶液NMR进行膜蛋白结构表征的重大进展奠定基础。为了接近膜蛋白，我们转向一种新的方法，在溶液NMR的封装蛋白质溶解在低粘度的流体。使用大部分螺旋KcsA钾通道和大部分β-折叠OmpA孔蛋白作为模型系统，我们将改进膜蛋白的反胶束包封方法。然后，我们将利用一个由500多种膜蛋白组成的卓越文库，该文库通过与von Heijne博士及其同事的合作提供给我们。超过一百种蛋白质以足够的水平表达，用于高通量生产和生物物理表征。我们将选择大约24个小的，高表达的膜蛋白，将其纳入反胶束和结构完整性测试。将确定表现最好的蛋白质的三维结构，验证在反胶束中将三重共振NMR方法应用于膜蛋白的可行性。收集成功封装的统计数据将有助于指导未来膜蛋白的高通量结构研究。

DESCRIPTION (provided by applicant): The ultimate goal of this proposal is to set the stage for significant advances in structural characterization of membrane proteins by solution NMR. To approach membrane proteins, we turn to a novel method in solution NMR of encapsulated proteins dissolved in low viscosity fluids. Using the largely helical KcsA potassium channel and the largely beta-sheet OmpA porin as model systems, we will refine the methods of reverse micelle encapsulation of membrane proteins. Then, we will take advantage of a remarkable library of more than 500 integral membrane proteins, which is available to us through collaboration with Dr. von Heijne and coworkers. Over one hundred proteins express at sufficient levels for high throughput production and biophysical characterization. We will select about two dozen of small, high-expressing membrane proteins that will be incorporated into reverse micelles and tested for structural integrity. Three dimensional structures of the best behaved protein(s) will be determined, validating the feasibility of applying triple resonance NMR methods in reverse micelles to membrane proteins. Gathered statistics of successful encapsulation will help guide future high-throughput structural studies of membrane proteins.