Structural studies of membrane proteins by NMR

描述(由申请人提供)：该提案的最终目标是为通过溶液核磁共振表征膜蛋白的结构特性方面的重大进展奠定基础。为了接近膜蛋白，我们转向了一种新的方法来研究溶解在低粘度流体中的微囊化蛋白质的溶液核磁共振。以大螺旋的KCSA钾通道和大β-折叠的OmpA孔蛋白为模型体系，进一步完善膜蛋白的反胶束包埋方法。然后，我们将利用一个非凡的文库，其中包含500多种完整的膜蛋白，这是我们通过与冯·海涅博士和同事的合作获得的。超过100种蛋白质的表达水平足以满足高通量生产和生物物理特性的需要。我们将选择大约24种小的、高表达的膜蛋白，这些蛋白将被掺入反胶束中并进行结构完整性测试。将确定表现最好的蛋白质(S)的三维结构，从而验证将反胶束中的三重共振核磁共振方法应用于膜蛋白质的可行性。收集的成功包膜的统计数据将有助于指导未来高通量的膜蛋白结构研究。

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.