Photodynamics and Maturation of Coral Fluorescent Proteins

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Time resolved crystallographic and kinetic studies will be used to investigate the unusual photocycle of amFP595, the "kindling fluorescent protein", a GFP-like chromoprotein isolated from reef organisms. The reversible light-induced transition from the dark nonfluorescent state to the fluorescent state and its subsequent slow relaxation in the dark will be analyzed at very high resolution using optical pumping and synchrotron radiation in various temperature regimes. The research will determine the nature of the structural transitions of the chromophore within the protein matrix, the nature of intermediate states and determine how the protein matrix controls both the ground state conformation and the excited state decay pathways of the chromophore. Mutagenesis combined with Arrhenius plot analysis will be used to study the energetic contributions of selected side chains to conformational stability, dark relaxation rates and reversibility of the fluorescence transition. These studies will be combined with limited crystallographic analysis and mutagenesis of two very closely related fluorescent proteins, blue fluorescent amFP486 and yellow fluorescent zFP538, in order to elucidate the structural basis for the differences in maturation behavior. It is postulated that the same chemical intermediates are formed in each protein, but suffer different final fates, due to the subtle effects of a small number of protein side chains close to the chromophore. While the research is directly relevant to the photodynamic processes involved in human vision, a potential practical outcome of the project is the development of a transient fluorescent tag for use in studies of material transport within living cells.Broader Impacts: The results of the proposed studies are expected to have broad impact as they will be relevant to the photophysics of many biological molecules, such as those involved in vision and photosynthesis. The results may also provide insight into the transient light/dark switching observed for small molecule and protein fluorophores used by many researchers. In the long term, the understanding gained from this work can be used to improve the properties of fluorescent proteins for use as tools for the study of the development and dynamic behavior of living creatures on both the subcellular and whole organism levels. The methodology will include a great variety of techniques drawn from physical chemistry and molecular biology and will thus provide excellent multidisciplinary training for graduate students and postdoctoral researchers. Due to their visual appeal, ease of production and isolation combined with exceptional physical stability, fluorescent proteins form an outstanding teaching resource for laboratory courses at the high school and university levels.