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

描述(申请人提供)：大约40%的人类基因组在细胞外围编码完整的膜蛋白，为细胞外信号与细胞内环境的交流提供必要的管道。这些蛋白质调节细胞内稳态的不同方面，作为细胞内信息传递的中心，包括营养吸收、能量生产、激素信号和药物外排机制，使癌细胞和病原体对其他有效的疾病治疗方法产生耐药性。以前的研究发现，迫切需要更深入地了解这些基本蛋白质的构象变化是如何指定它们的确切活性和信号功能的。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。这项研究的总体目标是通过开发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来满足这一需要，使用单分子荧光成像方法。完成拟议的目标将首次建立转运体动力学和摄取活性之间的定量关系。这也将为研究其他临床相关的小分子转运蛋白提供一个关键的基础，在这些转运蛋白中，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它们的活性。通过这项研究建立的技术基础也可以扩展到其他广泛的临床相关的、完整的膜蛋白，这些蛋白不会跨膜运输溶质。这些蛋白质包括G蛋白偶联受体(GPCRs)，其功能取决于触发下游信号级联的构象事件。如果成功，这些研究有可能从根本上改变在这一无处不在的蛋白质类别中可以实现的研究的性质、深度和广度。单分子成像技术基础的进步最终将使在活细胞中实时研究细胞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.