Optimizing and Analyzing Fluorescent Proteins

荧光蛋白（FP）已经彻底改变了生物医学研究，但这项技术的全部好处尚未实现。研究人员已经鉴定并设计了多种GFP相关蛋白，包括红色FP如DsRed，但许多新的FP比GFP发出更弱的荧光信号。此外，单体FP通常显示残余的自缔合，其导致荧光融合蛋白的错误定位甚至毒性。在这些问题得到解决之前，新的FP永远不会像GFP那样广泛使用。通过研究发色团形成的机制，将有助于FP的改进。 我们的目标是设计强大的定向进化策略，以提高亮度和最大限度地减少DsRed和其他FP的自关联。与此同时，野生型和突变型FP的结构-功能分析将有助于我们了解关键特性，如发色团成熟的途径。 该项目是两个具有互补专业知识的小组之间的合作。一个PI（Glick）已经广泛地工程化了DsRed，并且已经产生了分别在商业上称为“DsRed-Express”和“DsRed-Mexpress”的快速成熟的四聚体和单体变体。另一个PI（Keenan）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。这项建议有三个具体目标。 具体目标#1：开发用于产生较不容易自缔合的较亮FP的测定和程序。我们将使用同源性和结构引导的诱变来增强单体FP的荧光信号，并降低单体FP在高局部蛋白浓度下存在时在体内自缔合的趋势。 具体目标#2：设计用于测试单体FP是否是合适的融合标签的系统测定系统。我们将在酵母中将单体FP融合到一组96个潜在的聚集敏感性伴侣蛋白中。将对所得体内荧光图案进行详细的图像分析。 具体目标#3：表征DsRed的发色团成熟途径和其他性质。产生DsRed发色团的反应尚未完全了解。我们将测试一种新的成熟方案，可以解释以前令人困惑的结果。 荧光蛋白已成为基础和应用生物医学研究的关键实验工具。本文所述的研究将扩大这项技术的应用范围。

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.