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(GPCRs)代表了一大类高度同源的受体，它们具有共同的七个跨膜螺旋结构。这些受体通过接受细胞外刺激和激活细胞内G蛋白或阻滞素，构成了各种识别和信号通路的第一步。因此，对gpr结构和功能的了解具有很高的药学价值。GPCRs的结构相似之处表现在高度保守的氨基酸和连接的氢键网络的支架上。这些基序对于正常的受体功能也是必不可少的，因为它们的重组导致了伴随着受体激活的构象变化。这使得描绘GPCRs的一般结构/功能关系成为可能。视紫红质是脊椎动物眼睛视杆细胞中的光感受器，是一种非常适合机制研究的特殊GPCRs。视紫红质一直是众多生物物理和生化研究的对象，它的结构可以分解到氢键、单一氨基酸或内部水分子的水平。然而，即使对于视紫红质模型系统，构象动力学的描述也是非常有限的，尽管它们在受体功能中起着重要的作用。在本研究中，视紫红质以及其他GPCR将通过最近发展起来的位置定向自旋标记(SDSL)EPR技术来研究，该技术能够提供关于蛋白质构象平衡以及构象之间交换动力学的详细信息。通过使用压力调制，填充和描述功能上重要的构象是可行的，否则由于低密度而难以检测。利用压力跳跃和时间域EPR波谱，这些数据将得到蛋白质动力学信息的补充，以及这些动力学在受体功能期间是如何改变的。基于这些结果和对视紫红质原型的详细了解，将开发一个通用的GPCR激活的结构和功能框架。所尝试的通用模型的参数提供了定制的可能性，这是描述每种特定的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.