Energy landscape of GPCR-mediated G-protein activation by solution-state NMR

G蛋白偶联受体（GPCR）是细胞间信号转导途径的重要组成部分，因此是目前所有上市药物的约60%的靶点。尽管越来越多的晶体结构，GPCR介导的信号传导机制仍然没有得到充分的理解。一个原因可能是实验困难，以表征稀疏的激活状态，发生在信号使用X射线晶体学。在这个项目中，我们将使用溶液状态NMR光谱作为主要工具来表征GPCR与拮抗剂、激动剂或G蛋白结合后的结构和动力学变化。为了能够通过NMR研究这样的受体系统，我们使用了可以在E. coli，为尖端同位素标记提供了可能性。我们的初步数据表明，这种功能性GPCR代表了一个很好的模型系统的NMR为基础的研究的基本GPCR的活化过程中，到目前为止前所未有的质量和分辨率。在拟议项目的第二部分中，我们将研究由天然膜环境和与激活的GPCR结合诱导的G蛋白内的结构和动力学变化。我们选择的膜系统是正在实验室中开发用于NMR应用的磷脂纳米盘。我们将开发用于同位素标记的G蛋白的膜附着以及用于在天然脂质双层环境中与GPCR建立稳定复合物的生物化学方法。这些研究将提供GPCR介导的信号转导的结构和功能动力学的调查过程中发生的GPCR以及G蛋白的激活过程中稀疏的激活状态的重要信息。

G-protein coupled receptors (GPCRs) are essential components of the signal transduction pathways between cells and thus are targets of around 60% of all currently marketed drugs. Despite a growing number of crystal structures, the mechanism of GPCR-mediated signaling is still not sufficiently well understood. One reason might be experimental difficulties to characterize sparsely populated activated states that occur during signaling using X-ray crystallography. In this project, we will characterize the structural and dynamical changes in a GPCR upon binding to an antagonist, agonist or G-protein using solution-state NMR spectroscopy as a main tool. In order to be able to study such a receptor system by NMR, we use a signaling-competent and stabilized GPCR that can be produced in E. coli, providing the possibility for cutting-edge isotope labeling. Our preliminary data show that this functional GPCR represents an excellent model system for NMR-based studies of the essential GPCR activation processes in a so-far unprecedented quality and resolution. In the second part of the proposed project we will study the structural and dynamical changes within a G-protein that are induced by a native membrane environment and by binding to an activated GPCR. Our membrane system of choice are phospholipid nanodiscs that are being developed for NMR applications in the lab. We will develop biochemical methods for membrane attachment of an isotope-labeled G-protein as well as for the establishment of a stable complex with a GPCR in a native-like lipid bilayer environment. These studies will provide essential information on GPCR-mediated signal transduction by investigation of the structure and functional dynamics of sparsely populated activated states occurring during the activation process in GPCRs as well as G-proteins.