Microfluidics-based Selections for the Optimization of Red Flourescent Proteins

描述（由申请人提供）：该项目的总体目标是开发信号输出提高80倍的荧光蛋白（例如，光漂白前发射的光子数量）。在过去的10-15年里，荧光蛋白为细胞的基本工作提供了重要的见解，因为它们使研究人员能够可视化蛋白质运动，酶活性，基因表达，并实时量化活细胞中的重要信号分子。因此，荧光蛋白确实彻底改变了细胞生物学，揭示了细胞功能的基本生物学，同时帮助阐明了疾病状态中的错误。荧光蛋白信号输出的实质性改进需要下一个水平：单个活细胞内单个分子的可视化和监测。虽然在蛋白质工程领域已经有了一致的努力来提高荧光蛋白的特性，但最近的改进充其量是渐进的。我们假设，通过明确地设计荧光蛋白的发色团口袋和表面环境，可以获得80倍的信号输出改善。我们建议生成这些蛋白质的靶向文库，在哺乳动物细胞中表达这些文库，并使用我们最近实现的新型微流体细胞分选器筛选增加亮度、增加光稳定性和减少转化为“暗态”的蛋白质。此外，我们将进行多参数筛选，以确定增强多种光物理特性（例如亮度和光稳定性）的突变，并协同结合以大幅提高信号输出。这一信息是至关重要的，因为基于单一选择方案的蛋白质工程工作通常会以牺牲另一种特性为代价来优化一种特性，导致信号输出只有适度的增益。我们的目标是将序列多样性与功能多样性联系起来，以便深入了解荧光蛋白光物理特性的分子控制。这些信息不仅将用于未来的蛋白质设计工作，而且将帮助我们定义从荧光蛋白获得的最大信号输出。本研究有3个具体目标：(1)利用多相屏探测暗态弛豫率，使信号增加20倍；(2)利用光漂白探针使红色荧光蛋白的光稳定性提高至少7倍；(3)确定对光物理特性产生协同效应的突变组合。这将使我们能够将目标1和目标2中的增强功能与亮度的改进结合起来，从而使信号输出增加80倍。我们选择关注红色荧光蛋白，因为它们对单分子细胞成像的影响最大。新型荧光蛋白的开发与信号输出的大幅增加将极大地扩大我们的能力，以可视化和探测活细胞的内部工作。这些成像工具将通过提供对基本生物学和疾病进展机制的宝贵见解来影响公共卫生。

DESCRIPTION (provided by applicant): The broad goal of this project is to develop fluorescent proteins with an 80-fold improvement in signal output (e.g. number of photons emitted before photobleaching). Over the last 10-15 years, fluorescent proteins have provided critical insights into the fundamental workings of the cell as they enable researchers to visualize protein movements, enzyme activities, gene expression, and to quantify important signaling molecules in real time in living cells. As a result, fluorescent proteins have truly revolutionized cell biology, shedding light on the basic biology of cellular function, while helping to elucidate what goes wrong in disease states. Substantial improvements in the signal output of fluorescent proteins are required for the next level: visualizing and monitoring of single molecules within individual living cells. Although there has been a concerted effort in the protein engineering field to enhance fluorescent protein properties, recent improvements have been incremental at best. We hypothesize that an 80-fold improvement in signal output can be obtained by explicitly engineering both the chromophore pocket and surface environment of fluorescent proteins. We propose to generate targeted libraries of these proteins, express the libraries in mammalian cells, and screen proteins for increased brightness, increased photostability, and decreased conversion to "dark states" using a novel microfluidic cell sorter that we recently implemented. Moreover we will conduct multi-parameter screens in order to identify mutations that enhance multiple photophysical properties (for example brightness and photostability) and combine synergistically to improve signal output substantially. This information is crucial as protein engineering efforts based on a single selection scheme typically optimize one property at the expense of another, leading to only modest gains in signal output. Our goal is to connect sequence diversity to functional diversity in order to provide insight into the molecular control of photophysical properties in the fluorescent proteins. This information will not only be used in future protein design efforts, but will help us define the maximum signal output obtainable from fluorescent proteins. The proposed research has 3 Specific Aims: (1) To increase signal 20-fold with multi-phase screen that probes dark-state relaxation rate; (2) To increase photostability of red fluorescent proteins by at least 7-fold with screen that probes photobleaching; (3) To identify combinations of mutations that yield synergistic effects on photophysical properties. This will enable us to combine enhancements identified in aim 1 and 2, with improvements in brightness to yield an 80- fold increase in signal output. We have chosen to focus on red fluorescent proteins because these have the greatest potential for impacting single molecule cellular imaging. The development of new fluorescent proteins with substantial increases in signal output will dramatically expand our ability to visualize and probe the inner workings of living cells. These imaging tools will impact public health by providing valuable insight into basic biology and mechanisms of disease progression.