Developing a common structural and functional framework for G Protein coupled receptors by means of site-directed spin labeling and pressure modulation EPR spectroscopy

G蛋白偶联受体（gpcr）是一大群高度同源的受体，它们具有共同的7个跨膜螺旋结构。这些受体通过接受细胞外刺激和激活细胞内G蛋白或阻滞蛋白，构成各种识别和信号通路的第一步。因此，了解GPCR的结构和功能具有很高的药学价值。gpcr具有高度保守的氨基酸和连接的氢键网络。这些基序对于受体的正常功能也是必不可少的，因为它们的重组会导致伴随受体激活的构象变化。这使得描述gpcr的一般结构/功能关系成为可能。一种特殊的GPCR非常适合于机制研究，它是视紫红质，脊椎动物眼睛杆状细胞中的光受体。紫红质已成为许多生物物理和生化研究的主题，将其结构分解为氢键，单氨基酸或内部水分子的水平。然而，构象动力学已经描述到非常有限的程度，甚至对视紫红质模型系统，尽管他们的受体功能的重要作用。在本研究中，视紫红质和其他gpcr将通过最近开发的位点定向自旋标记（SDSL） EPR技术进行研究，该技术能够提供蛋白质构象平衡的详细信息以及构象之间交换的动态。通过使用压力调制，可以填充和描述功能重要的构象，否则由于低种群而难以检测。利用压力跳变和时域EPR光谱，这些数据将被蛋白质动力学信息以及这些动力学在受体功能过程中如何改变的信息所补充。基于研究结果和对视紫红质原型的详细了解，将开发GPCR激活的共同结构和功能框架。尝试通用模型的参数提供了定制的可能性，这是描述每种特定GPCR类型所需的。

G protein coupled receptors (GPCRs) represent a large group of highly homologous receptors which share a common seven transmembrane helical architecture. These receptors constitute the first step of various recognition and signaling pathways by accepting extracellular stimuli and activating intracellular G proteins or arrestins. Knowledge of GPCR structure and function is therefore of high pharmaceutical interest. The close structural similarity of GPCRs is manifested by a scaffold of highly-conserved amino acids and connecting hydrogen bond networks. These motifs are also indispensable for proper receptor function as their reorganization renders conformational changes which accompany receptor activation. This makes it is feasible to delineate general structure/function relations for GPCRs.One specific GPCR which is well suited for mechanistic studies is rhodopsin, the light receptor in the rod cell of the vertebrate eye. Rhodopsin has been subject of numerous biophysical and biochemical studies resolving its structure down to the level of hydrogen bonds, single amino acids or internal water molecules. However, conformational dynamics have been described to a very limited extent even for the rhodopsin model system, despite their important role for receptor function.In this study rhodopsin as well as other GPCRs will be investigated by recently developed site-directed spin labeling (SDSL) EPR techniques which are capable of providing detailed information on protein conformational equilibria as well as the dynamics of exchange between conformations. By using pressure modulation it is feasible to populate and describe functionally important conformations which are otherwise difficult to detect due to low populations. Using pressure-jump and time domain EPR spectroscopy, these data will be complemented by information on protein dynamics and how these dynamics are altered during receptor function. Based on the results and the detailed knowledge of the rhodopsin archetype, a common structural and functional framework of GPCR activation will be developed. Parameters of the attempted universal model provide the possibility of customization which is needed to describe each specific GPCR types.