Probing Downhill Folding using Microfluidics and Single-molecule Fluorescence

在这项提案中，PI将研究蛋白质如何在活跃的三维结构中折叠。根据蛋白质折叠的景观理论得出的一个基本预测是，蛋白质折叠可以在多维折叠漏斗上快速下坡进行。在这个项目中，PI将使用单分子荧光(SMF)和微流控方法的组合来更直接地探索下坡折叠。该项目将重点关注以下具体目标。1)使用超快微流控混合器和集成荧光技术测量多个可能的下坡折叠蛋白的折叠/去折叠速率。2)直接探索单个蛋白质分子折叠的下坡性。微流控混合将触发折叠/展开，SMF将用于跟踪过渡过程中的种群分布和动态。将为这些实验改进非平衡SMF/微流控方法，特别是在时间分辨率和触发方面。3)为了进一步提高触发的时间分辨率，PI还将探索光引发捕获在新的微流控设备中的单个蛋白质分子折叠的方法。拟议活动的主要学术价值是提供了对这一迄今难以捉摸的过程的第一次直接观察，并对蛋白质折叠的景观理论进行了实质性的测试。这些实验还将被用来更好地研究折叠能量景观的许多迄今隐藏的细节。在项目期间开发的微流体/SMF组合方法将对需要解决快速非平衡动力学的广泛的单分子研究有用，因此将对科学界具有普遍意义。该项目的工具和一般见解将得到广泛传播。本科生和博士后将在这个跨学科项目的过程中接受不断发展的最先进技术的培训，该项目涉及两个机构和来自物理、生物和化学的组成部分。该项目的结果也将被纳入课堂。

In this proposal the PIs will study how proteins fold in an active 3-dimensional structure. A fundamental prediction following from the landscape theory of protein folding is that protein folding can occur rapidly downhill on a multidimensional folding funnel. In this project the PIs will use a combination of single molecule fluorescence (SMF) and microfluidic methods to more directly explore downhill folding. The project will focus on the following specific goals. 1) Measure folding/unfolding rates of multiple putative downhill folding proteins using an ultrafast microfluidic mixer and ensemble fluorescence. 2) Directly probe the downhill character of folding for single protein molecules. Microfluidic mixing will trigger the folding/unfolding, and SMF will be used to follow the distribution of populations and dynamics during the transition. Non-equilibrium SMF/microfluidics methods, specifically with respect to time resolution and triggering will be improved for these experiments. 3) In order to further improve the time-resolution of the triggering, the PIs will also explore methods to photo-initiate folding of single protein molecules trapped in a new microfluidic device. The main intellectual merit of the proposed activity is to provide the first direct observations of this so-far elusive process, and provide a substantial test of the landscape theory of protein folding. These experiments will also be used to better examine many so-far hidden details of folding energy landscapes. The combined microfluidics/SMF methods developed during the project will be useful for a wide range of single-molecule investigations where the fast non-equilibrium kinetics needs to be resolved, and thus will be of general utility to the scientific community. The tools and general insights from this project will be disseminated widely. Undergraduate and post-doctoral students will receive training in evolving state-of-the-art technologies during the course of this interdisciplinary project involving two institutions and components from physics, biology and chemistry. Results from the project will also be incorporated into classes.