Probing Downhill Folding using Microfluidics and Single-molecule Fluorescence

使用微流体和单分子荧光探测下坡折叠

• 批准号: 0750049
• 负责人: Ashok Deniz
• 金额: $ 43.5万
• 依托单位: The Scripps Research Institute
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0750049&HistoricalAwards=false
• 关键词: Probing; Downhill; Folding; using; Microfluidics

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.

在该提案中，PI 将研究蛋白质如何在活跃的 3 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向下发生。在这个项目中，PI 将结合使用单分子荧光 (SMF) 和微流体方法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) 使用超快微流体混合器和整体荧光测量多种假定的下坡折叠蛋白的折叠/解折叠速率。 2）直接探测单个蛋白质分子折叠的下坡特征。微流体混合将触发折叠/展开，SMF 将用于跟踪过渡期间的群体分布和动态。非平衡 SMF/微流体方法，特别是在时间分辨率和触发方面，将针对这些实验进行改进。 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型微流体装置中的单个蛋白质分子折叠的方法。该活动的主要智力价值是提供对这一迄今为止难以捉摸的过程的首次直接观察，并对蛋白质折叠的景观理论进行实质性测试。这些实验还将用于更好地检查折叠能源景观的许多迄今为止隐藏的细节。该项目期间开发的微流体/SMF 组合方法将适用于需要解决快速非平衡动力学的各种单分子研究，因此对科学界具有普遍实用性。该项目的工具和一般见解将得到广泛传播。在这个跨学科项目的过程中，本科生和博士后学生将接受不断发展的最先进技术的培训，该项目涉及物理、生物和化学的两个机构和组成部分。该项目的结果也将纳入课堂。