Towards a structural and temporal understanding of phototransduction

描述（由申请人提供）：G蛋白偶联受体（GPCR）介导的信号传导系统的潜在机制仍然没有解决，尽管这些系统在过去的世纪受到了强烈关注。这部分是由于在研究膜蛋白在其自然环境中的方法，提供分子细节的困难。目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的奥秘。视紫红质和视觉系统将是该研究计划的最初重点。这种原型GPCR信号系统提供了几个优点，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，这些图像将提供系统的结构和组织信息。单分子力光谱将提供有关视紫红质中稳定蛋白质并促进其功能的分子相互作用的详细信息。冷冻电子断层扫描将获得一个未受干扰的杆外节，提供结构信息，这个隔间和大分子，在这个地点进行他们的功能。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，以监测定义信号传导过程的动态相互作用以及发生这种相互作用的时间范围。通过这种独特的方法组合获得的信息将提供这些系统目前无法获得的关键分子信息。这将有助于定义信号事件背后的分子机制，这些信号事件支配由GPCR调控的重要生理过程。 G蛋白偶联受体（GPCR）代表了目前市场上最大的一类细胞表面蛋白和药物靶点。这个蛋白质家族几乎参与了所有的生理过程，这些系统的功能障碍可能导致失明、成瘾、糖尿病和心脏病等疾病。尽管GPCR的重要性，其作用的准确分子描述仍然缺乏。 了解这些系统的分子奥秘将有助于开发更有效的治疗方案。

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.