Structure-Function in Red and Yellow Fluorescent Proteins

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，这些同源物可发出红光（drFP583）、黄光（zFP538）或有色但不荧光（asFP595）。目的是发现导致这些分子荧光的独特化学和三维构型。基于从这项工作中获得的理解，从长远来看，这些蛋白质的性质将得到改善，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的发光研究工具。一般的方法将包括晶体学、物理化学和定向诱变研究。一级氨基酸序列与三级和四级结构之间的关系将由高分辨率x射线晶体学确定。特定侧链在成熟化学中的作用将通过定向诱变和变体的光吸收和发射特性分析来确定。选择突变体，以优化发射颜色，寡聚化状态和分子对外部条件的响应，用于在活细胞中实时作为溶液条件（例如pH）的视觉指标。最终的结果将是为研究界提供一种工具，以无创的方式报告基因表达的时间、基因产物的定位和细胞化学的变化。在大多数情况下，随着这项工作的进行，研究生将获得一些跨学科领域的培训。

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.