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

通过定点自旋标记和压力调制 EPR 光谱法开发 G 蛋白偶联受体的通用结构和功能框架

• 批准号: 254065586
• 负责人: Dr. Matthias Elgeti
• 金额: --
• 依托单位: Institut für Medizinische Physik und Biophysik
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/254065586?language=en
• 关键词: Developing; common; structural; functional; framework

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.

G蛋白偶联受体（GPCR）代表了一大组高度同源的受体，其共享共同的七跨膜螺旋结构。这些受体通过接受细胞外刺激和激活细胞内G蛋白或抑制蛋白，构成各种识别和信号传导途径的第一步。因此，GPCR结构和功能的知识具有很高的药学兴趣。GPCR的紧密结构相似性通过高度保守的氨基酸的支架和连接氢键网络来表现。这些基序对于适当的受体功能也是必不可少的，因为它们的重组使得伴随受体活化的构象变化。这使得它是可行的，描绘一般的结构/功能关系的GPCRs。一个特定的GPCR，这是非常适合于机制的研究是视紫红质，光受体在视杆细胞的脊椎动物的眼睛。视紫红质已经成为许多生物物理和生物化学研究的主题，将其结构解析到氢键，单个氨基酸或内部水分子的水平。然而，构象动力学已被描述到一个非常有限的程度，即使是视紫红质模型系统，尽管他们的重要作用受体function.In这项研究视紫红质以及其他GPCR将调查最近开发的定点自旋标记（SDSL）EPR技术，能够提供详细的信息，蛋白质构象平衡以及构象之间的交换动力学。通过使用压力调制，它是可行的，以填充和描述功能上重要的构象，否则难以检测由于低人口。使用压力跳跃和时域EPR光谱，这些数据将补充蛋白质动力学的信息，以及这些动力学是如何在受体功能的改变。基于结果和视紫红质原型的详细知识，将开发GPCR激活的共同结构和功能框架。所尝试的通用模型的参数提供了定制的可能性，这是描述每种具体的气相化学还原所需要的。