Towards a structural and temporal understanding of phototransduction

描述（由申请人提供）：尽管在过去的一个世纪里这些系统受到了广泛关注，但G蛋白偶联受体（GPCR）介导的信号系统的机制仍未解决。这部分是由于通过提供分子细节的方法在其天然环境中研究膜蛋白的困难。当前建议的重点是应用新的生物物理方法来解开gpcr介导的信号通路的分子和时间奥秘。视紫红质和视觉系统将是研究计划的最初重点。这种原型GPCR信号系统提供了几个优势，将允许应用新的生物物理方法。原子力显微镜将产生单个分子的高分辨率图像，将提供系统的结构和组织信息。单分子力谱将提供紫红质中稳定蛋白质和促进其功能的分子相互作用的详细信息。低温电子断层扫描将获得一个未受扰动的棒外段，以提供有关该隔室和在该场所执行其功能的大分子的结构信息。荧光共振能量转移将用于检测信号蛋白的蛋白质-蛋白质相互作用，以监测定义信号过程和发生时间框架的动态相互作用。通过这种独特的方法组合获得的信息将提供目前这些系统无法获得的关键分子信息。这将有助于确定由gpcr调控的重要生理过程的信号事件的分子机制。

DESCRIPTION (provided by applicant): The mechanism underlying G protein-coupled receptor (GPCR)-mediated signaling systems is still unresolved despite the intense focus these systems have received over the past century. This is due in part to the difficulties in studying membrane proteins in their native context by methods that provide molecular details. The focus of the current proposal is to apply novel biophysical methodologies to unravel the molecular and temporal mysteries of GPCR-mediated signaling pathways. Rhodopsin and the visual system will be the initial focus of the research program. This prototypical GPCR signaling system offers several advantages that will allow for the application of novel biophysical approaches. Atomic force microscopy will result in high-resolution images of individual molecules that will provide structural and organizational information of the system. Single-molecule force spectroscopy will provide detailed information on the molecular interactions in rhodopsin that stabilize the protein and promote its function. Cryo-electron tomography will gain access to an unperturbed rod outer segment to provide structural information on this compartment and on the macromolecules that carry out their function at this venue. Fluorescence resonance energy transfer will be utilized to detect protein-protein interactions of signaling proteins to monitor the dynamic interactions that define the signaling process and the timeframe in which this takes place. Together the information obtained by this unique combination of methodologies will provide key pieces of molecular information that is currently unavailable for these systems. This will help define the molecular mechanism underlying the signaling events that govern important physiological processes regulated by GPCRs. G protein-coupled receptors (GPCRs) represent the largest class of cell surface proteins and drug targets currently on the market. This family of proteins is involved in virtually every physiological process, and dysfunctions in these systems can lead to diseases such as blindness, addiction, diabetes, and heart disease. Despite the importance of GPCRs an accurate molecular description of their action is still lacking. Understanding the molecular mysteries of these systems will lead to the development of more effective therapeutic solutions.