Structure-Function in Red and Yellow Fluorescent Proteins

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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.