Probing Downhill Folding using Microfluidics and Single-molecule Fluorescence

在这个提议中，pi将研究蛋白质如何在一个活跃的三维结构中折叠。蛋白质折叠景观理论的一个基本预测是，蛋白质折叠可以在多维折叠漏斗上快速下坡。在这个项目中，pi将使用单分子荧光（SMF）和微流体方法的组合来更直接地探索下坡折叠。该项目将侧重于以下具体目标。1)使用超快微流体混合器和集合荧光测量多种假定的下端折叠蛋白的折叠/展开速率。2)直接探测单个蛋白质分子折叠的下坡特性。微流体混合将触发折叠/展开，SMF将用于跟踪过渡期间的种群分布和动力学。非平衡SMF/微流体方法，特别是时间分辨率和触发将在这些实验中得到改进。3)为了进一步提高触发的时间分辨率，研究人员还将探索一种新的微流体装置中捕获的单蛋白分子的光引发折叠方法。这项活动的主要学术价值在于提供了对这一迄今为止难以捉摸的过程的第一次直接观察，并为蛋白质折叠的景观理论提供了实质性的测试。这些实验还将用于更好地研究迄今为止隐藏的折叠能量格局的许多细节。在该项目中开发的微流体/SMF联合方法将用于需要解决快速非平衡动力学的大范围单分子研究，因此将对科学界具有普遍效用。这个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涉及物理、生物和化学两个机构和组成部分的跨学科项目中接受发展最先进技术的培训。该项目的结果也将纳入课堂。

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.