Optimizing and Analyzing Fluorescent Proteins

荧光蛋白的优化和分析

• 批准号: 7822753
• 负责人: BENJAMIN S GLICK
• 金额: $ 23.53万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-15 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7822753
• 关键词: Amino Acid Substitution; Biological Assay; Biomedical Research; Chimeric Proteins; Collaborations; DsRed; Engineering; Environment; Fluorescence; Goals; Image Analysis; Methods; Mutagenesis; Pathway interactions; Pattern; Photobleaching; Procedures; Property; Protein Family; Proteins; Reaction; Research Personnel; Residual state; Scheme; Signal Transduction; Simulate; Structural Biologist; Structure; System; Technology; Testing; Toxic effect; Variant; Yeasts; chromophore; directed evolution; improved; in vivo; monomer; mutant; novel; quantum; red fluorescent protein; tool

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.

描述（由申请人提供）：荧光蛋白（FPS）彻底改变了生物医学研究，但该技术的全部好处尚未实现。研究人员已经确定并设计了各种与GFP相关的蛋白质，包括红色FPS（例如DSRED），但其中许多新FPS比GFP弱荧光信号。此外，单体FPS经常显示出残留的自我关联，导致荧光融合蛋白的毒性不足甚至毒性。在修复这些问题之前，新的FPS永远不会像GFP那样广泛有用。通过研究发色团形成的机制，将有助于FPS的改善。 我们的目标是制定强大的定向进化策略，以增强亮度并最大程度地减少DSRED和其他FPS的自我关联。同时，野生型和突变体FPS的结构功能分析将有助于我们了解关键特性，例如发色团成熟的途径。 该项目是具有互补专业知识的两个小组之间的合作。一个PI（Glick）已进行了广泛设计的DSRED，并分别创建了商业上称为“ DSRED-EXPRESS”和“ DSRED-MENOMER”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，也是通过定向进化来改善蛋白质的专家。该提案具有三个具体目标。 特定目的＃1：开发生成较亮FP的测定法和程序，这些FPS不易自我关联。我们将使用同源性和结构引导的诱变来增强单体FPS的荧光信号，并降低以高局部蛋白质浓度存在时单体FPS在体内自我关联的趋势。 特定目的＃2：设计一个系统的测定系统，用于测试单体FP是否是合适的融合标签。我们将单体FP融合到酵母中的一组96个潜在的敏感伴侣蛋白。由此产生的体内荧光模式将进行详细的图像分析。 特定目的＃3：表征发色团成熟途径和DSRED的其他特性。产生DSRED发色团的反应未完全理解。我们将测试一种新型的成熟方案，该方案可以解释先前令人困惑的结果。 荧光蛋白已成为基本和应用生物医学研究的关键实验工具。这里描述的研究将扩大该技术的应用。

项目成果

A rapidly maturing far-red derivative of DsRed-Express2 for whole-cell labeling.

• DOI: 10.1021/bi900870u
• 发表时间: 2009-09-08
• 期刊: BIOCHEMISTRY
• 影响因子: 2.9
• 作者: Strack, Rita L.;Hein, Birka;Bhattacharyya, Dibyendu;Hell, Stefan W.;Keenan, Robert J.;Glick, Benjamin S.
• 通讯作者: Glick, Benjamin S.

Noncytotoxic orange and red/green derivatives of DsRed-Express2 for whole-cell labeling.

• DOI: 10.1186/1472-6750-9-32
• 发表时间: 2009-04-03
• 期刊: BMC biotechnology
• 影响因子: 3.5
• 作者: Strack RL;Bhattacharyya D;Glick BS;Keenan RJ
• 通讯作者: Keenan RJ

Noncytotoxic DsRed derivatives for whole-cell labeling.

• DOI: 10.1007/978-1-61737-950-5_17
• 发表时间: 2011
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Strack, Rita L;Keenan, Robert J;Glick, Benjamin S
• 通讯作者: Glick, Benjamin S

Chromophore formation in DsRed occurs by a branched pathway.

• DOI: 10.1021/ja1030084
• 发表时间: 2010-06-23
• 期刊: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
• 影响因子: 15
• 作者: Strack, Rita L.;Strongin, Daniel E.;Mets, Laurens;Glick, Benjamin S.;Keenan, Robert J.
• 通讯作者: Keenan, Robert J.