Quantitative investigations of transporter dynamics and uptake at the single-mole

描述（由申请人提供）：大约40%的人类基因组编码细胞外周的完整膜蛋白，为细胞外信号与细胞内环境的通信提供必要的通道。这些蛋白介导细胞内稳态的各个方面，作为细胞内信息传递的中心枢纽，包括营养摄取、能量产生、激素信号和药物外排机制，这些机制使癌细胞和病原体对其他有效的疾病治疗产生抗性。先前的研究表明，迫切需要更深入地了解这些基本蛋白质的构象变化如何指定它们的精确活动和信号功能。因此，迫切需要新的生物物理方法，能够从运动的角度直接测量膜蛋白的活性。提出的研究的总体目标是通过发展一种通用的方法来直接确定膜蛋白动力学和活性之间的关系，使用单分子荧光成像方法来满足这一需求。拟议目标的完成将首次建立转运体动力学和摄取活性之间的定量关系。它还将为研究其他临床相关的小分子转运蛋白提供重要的基础，其中需要更深入地了解膜蛋白动力学和活性之间的关系，以了解小分子如何调节其活性。通过这项研究建立的技术基础也可以扩展到广泛的其他临床相关的、不跨膜运输溶质的整体膜蛋白。这些蛋白质包括G蛋白偶联受体（gpcr），其功能取决于触发下游信号级联的构象事件。如果成功的话，这些研究有可能从根本上改变研究的性质、深度和广度，这些研究可以在这种无处不在的蛋白质类别中实现。单分子成像技术基础的进步最终将使研究活细胞中膜上的信号事件成为可能。

DESCRIPTION (provided by applicant): Roughly forty percent of the human genome encodes integral membrane proteins at the cell periphery that provide essential conduits for the communication of extracellular signals to the intracellular milieu. These proteins mediate diverse aspects of cellular homeostasis, serving as central hubs for information transfer within the cell, including nutrient uptake, energy production, hormone signaling, and drug efflux mechanisms that render cancer cells and pathogens resistant to otherwise effective therapeutic treatments of disease. Insights from prior investigations have revealed a pressing need for a deeper understanding of how conformational changes in these essential proteins specify their precise activities and signaling functions. Consequently, there is an urgent demand for new biophysical approaches that enable direct measurements of membrane protein activities from the perspective of motion. The overarching goal of the proposed research is to meet this need through the development of a generalizable approach to directly ascertain the relationship between membrane protein dynamics and activity using single-molecule fluorescence imaging methods. Completion of the proposed Aims will establish a quantitative relationship between transporter dynamics and uptake activity for the first time. It will also provide a critical foundaion for investigating other clinically relevant small-molecule transporters, where a deeper understanding of the relationship between membrane protein dynamics and activity is required to understand how small-molecules modulate their activities. The technological foundations established through this research may also be extended to a broad range of other clinically relevant, integral membrane proteins that do not transport solutes across the membrane. Such proteins include G protein coupled receptors (GPCRs) whose functions hinge upon conformational events that trigger downstream signaling cascades. If successfully enabled, these investigations have the potential to fundamentally change the nature, depth and breadth of investigations that can be achieved within this ubiquitous protein class. Advancements in the technological foundations of single-molecule imaging will ultimately enable investigations of signaling events at the membrane in real time in living cells.