Photodynamics of Fluorescent Proteins

University Of Oregon Eugene, Eugene OR

Investigators

Abstract

mTFP0.7 is a monomeric, bright cyan fluorescent protein that nevertheless has high sequence and structural homology with the red and yellow reef GFPs. Static and time-resolved X-ray crystallography, ultrafast spectroscopy and mutational analysis will be used to investigate the remarkable photodynamic behavior of mTFP0.7. This protein undergoes facile photoswitching between fluorescent and nonfluorescent states while the derived but closely related protein, mTPF1, is photostable. Spectroscopic, thermodynamic and comparative structural studies will help understand how the three dimensional structures of the proteins control the emission properties of the internal chromophore. The time resolved structural and spectroscopic studies of mTFP0.7 will reveal details of molecular rearrangements, due to chromophore isomerization, that are broadly relevant to the photophysics of many biological molecules (such as those involved in vision and photosynthesis) and will provide insight into the nature of transient light/dark switching processes observed for other types of fluorophores. The understanding gained from this work will be useful to improve the properties of these and related fluorescent proteins for use as practical, light emitting probes for studies of living cells. Anticipated uses of photoactivatable probes, also termed optical highlighters, include selective marking of molecular assemblies for study of active transport within cells and development of new techniques in high resolution fluorescence microscopy. Broader Impacts The interdisciplinary nature of the effort and the great variety of experimental techniques will provide excellent multidisciplinary training for students and postdoctoral researchers. Due to their visual appeal, ease of production and isolation combined with exceptional physical stability, fluorescent proteins provide outstanding teaching material for laboratory courses at the high school and university levels and the results from this research will be used for the development of new instructional protocols. Finally, animated illustrations of observed molecular motions and other details of the photodynamic behavior will be disseminated to the interested public via the Internet, in the form of instructional videos and textual materials.