Structure-Function in Red and Yellow Fluorescent Proteins

红色和黄色荧光蛋白的结构-功能

• 批准号: 0111053
• 负责人: Stephen Remington
• 金额: $ 37.5万
• 依托单位: University of Oregon Eugene
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0111053&HistoricalAwards=false
• 关键词: Structure; Function; Red; Yellow; Fluorescent

The purpose of this investigation will be to determine the structural basis for the maturation chemistry and fluorescent properties of three newly discovered green fluorescent protein homologies (Fps) which emit red light (drFP583), yellow light (zFP538) or are colored but not fluorescent (asFP595). The objective is to discover the unique chemistry and three dimensional configurations that lead to the fluorescence of these molecules. On the basis of the understanding gained form this work, in the long term the properties of these proteins will be improved for use as light emitting research tools for the study of the development and dynamic behavior of living creatures on both the subcellular and whole organism levels.The general approach will include crystallographic, physicochemical and directed mutagenesis studies. The relationships between the primary amino acid sequences and the tertiary and quaternary structures will be determined by high resolution X-ray crystallography. The roles of specific side chains in the maturation chemistry will be determined by directed mutagenesis and analysis of the light absorption and emission properties of the variants. Selected mutants will be made in order to optimize emission color, state of oligomerization and molecular response to external condition for use as visual indicators of solution conditions (e.g. pH) in real time in living cells. The end result will be to provide the research community with tools that report the timing of gene expression, localization of gene products, and changes in cellular chemistry in a noninvasive manner. For the most part, graduate students will gain training in a number of interdisciplinary areas as this work proceeds.

本研究的目的是确定三种新发现的绿色荧光蛋白同源物（Fps）的成熟化学和荧光特性的结构基础，这些Fps发出红光（drFP 583）、黄光（zFP 538）或有色但不发荧光（asFP 595）。 目的是发现导致这些分子荧光的独特化学和三维构型。 在此基础上，从长远来看，这些蛋白质的性质将得到改善，作为发光研究工具，用于在亚细胞和整个生物体水平上研究生物的发育和动态行为，一般的方法包括晶体学、物理化学和定向诱变研究。 将通过高分辨率X射线晶体学确定一级氨基酸序列与三级和四级结构之间的关系。 特定侧链在成熟化学中的作用将通过定向诱变和分析变体的光吸收和发射性质来确定。 将制备选择的突变体，以优化发射颜色、寡聚化状态和对外部条件的分子响应，用作活细胞中真实的溶液条件（例如pH）的视觉指示剂。 最终的结果将是为研究界提供以非侵入性方式报告基因表达时间、基因产物定位和细胞化学变化的工具。 在大多数情况下，研究生将获得培训，在一些跨学科领域的这项工作的进展。