Microfluidics-based Selections for the Optimization of Red Fluorescent Proteins

描述（由申请人提供）：这是对GM083849“基于微流体的红色荧光蛋白优化选择”的竞争性修订。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。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。这次修订将在原来的三个目标的基础上增加一个新的具体目标。新目标的两个主要目标是：1)利用生物物理数据指导红色荧光蛋白（rfp）的文库设计；2)提供改善的光物理性质与蛋白质结构和动力学变化之间的机制联系。这个新的目标扩展了原来的目标，使用最先进的核磁共振（NMR）技术来收集RFP突变体的原子水平结构和动态数据，这将加速开发改进的荧光蛋白的总体目标。这些数据将用于开发结构、动力学和光物理性质（光稳定性、暗态转换和亮度）变化之间的相关性。我们建议结合核磁共振方法来研究肽的主链和侧链结构和动力学。这些信息将定义图书馆设计中针对的局部构象灵活性区域。这将为文库设计提供一个更明智的起点，并简化我们产生改进的荧光蛋白的努力，从而加速实现最初提议的目标。

DESCRIPTION (provided by applicant): This is a competitive revision of the grant GM083849 "Microfluidics-based selections for the optimization of red fluorescent proteins." 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. 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. This revision will add a new specific aim to the original three aims. The two main goals of the new aim are: i) to use biophysical data to guide library design of red fluorescent proteins (RFPs) and ii) to provide a mechanistic link between improved photophysical properties and changes in protein structure and dynamics. This new aim expands upon the original aims by using state-of-the-art nuclear magnetic resonance (NMR) techniques to collect atomic level structural and dynamic data for RFP mutants, which will accelerate the overall goal of developing improved fluorescent proteins. These data will be used to develop correlations between changes in structure and dynamics and photophysical properties (photostability, dark-state conversion, and brightness). We propose a combination of NMR methodologies to investigate peptide backbone and side chain structure and dynamics. This information will define regions of local conformational flexibility to be targeted in library designs. This will provide a more informed starting point for library design and streamline our efforts to generate improved fluorescent proteins and thus will accelerate the goals of the original proposal. PUBLIC HEALTH RELEVANCE: 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.