Optimizing and Analyzing Fluorescent Proteins

描述(由申请人提供)：荧光蛋白(FP)使生物医学研究发生了革命性的变化，但这项技术的全部好处尚未实现。研究人员已经识别并改造了各种与GFP相关的蛋白质，包括DsRed等红色FP，但这些新FP中的许多发出的荧光信号比GFP弱。此外，单体FP往往表现出残留的自结合，导致荧光融合蛋白的错误定位甚至毒性。除非这些问题得到解决，否则新的FP永远不会像GFP那样广泛使用。通过研究生色团的形成机制，将有助于FP的改进。 我们的目标是设计稳健的定向进化策略，以增强DsRed和其他FP的亮度并将其自关联降至最低。同时，对野生型和突变型FP的结构-功能分析将有助于我们了解发色团成熟的途径等关键性质。 这个项目是两个具有互补专业知识的小组之间的合作。One PI(Glick)对DsRed进行了广泛的工程设计，并创造了快速成熟的四聚和单体变体，分别被商业上称为“DsRed-Express”和“DsRed-Monomer”。另一位派(基南)是一位熟练的结构生物学家，也是通过定向进化改进蛋白质的专家。这项提议有三个具体目标。 具体目标#1：开发产生更明亮的FP的分析和程序，这些FP不太容易自我关联。我们将使用同源性和结构导向的突变来增强单体FP的荧光信号，并降低单体FP在高局部蛋白质浓度下在体内自结合的倾向。 具体目的#2：设计一种系统的检测系统，用于检测单体FP是否为合适的融合标签。我们将在酵母中将一个单体FP融合到一组96个潜在聚集敏感的伙伴蛋白上。由此产生的体内荧光图案将接受详细的图像分析。 特定目标#3：表征DsRed的发色团成熟途径和其他性质。产生DsRed发色团的反应还不完全清楚。我们将测试一种新的成熟方案，该方案可以解释之前令人困惑的结果。 荧光蛋白已成为基础和应用生物医学研究的重要实验工具。本文所述的研究将扩大该技术的应用范围。

DESCRIPTION (provided by applicant): Fluorescent proteins (FPs) have revolutionized biomedical research, but the full benefits of this technology have yet to be realized. Researchers have identified and engineered a variety of GFP-related proteins, including red FPs such as DsRed, but many of these new FPs give weaker fluorescence signals than GFP. Moreover, monomeric FPs often show a residual self- association that results in mislocalization and even toxicity of fluorescent fusion proteins. Until these problems are remedied, the new FPs will never be as broadly useful as GFP. The improvement of FPs will be aided by investigating the mechanisms of chromophore formation. Our goal is to devise robust directed evolution strategies for enhancing the brightness and minimizing the self-association of DsRed and other FPs. In parallel, structure-function analysis of wild-type and mutant FPs will help us to understand key properties such as the pathway of chromophore maturation. This project is a collaboration between two groups with complementary expertise. One PI (Glick) has extensively engineered DsRed, and has created rapidly maturing tetrameric and monomeric variants known commercially as "DsRed-Express" and "DsRed-Monomer", respectively. The other PI (Keenan) is a skilled structural biologist and an expert at improving proteins by directed evolution. This proposal has three Specific Aims. Specific Aim #1: To develop assays and procedures for generating brighter FPs that are less prone to self-association. We will use homology- and structure-guided mutagenesis to enhance the fluorescence signals from monomeric FPs, and to reduce the tendency of monomeric FPs to self-associate in vivo when present at high local protein concentrations. Specific Aim #2: To devise a systematic assay system for testing whether a monomeric FP is a suitable fusion tag. We will fuse a monomeric FP to a set of 96 potentially aggregation- sensitive partner proteins in yeast. The resulting in vivo fluorescence patterns will be subjected to detailed image analysis. Specific Aim #3: To characterize the chromophore maturation pathway and other properties of DsRed. The reactions that generate the DsRed chromophore are incompletely understood. We will test a novel maturation scheme that can explain previously puzzling results. Fluorescent proteins have become key experimental tools for basic and applied biomedical research. The studies described here will expand the applications of this technology.