Photodynamics of Fluorescent Proteins

mTFP0.7是一种单体的亮青色荧光蛋白，但与红色和黄色珊瑚礁GFP具有高度的序列和结构同源性。静态和时间分辨的X射线晶体学，超快光谱和突变分析将被用来研究显着的mTFP0.7的光动力学行为。这种蛋白质在荧光和非荧光状态之间进行了简单的光开关，而衍生的但密切相关的蛋白质mTPF 1是光稳定的。光谱、热力学和比较结构研究将有助于理解蛋白质的三维结构如何控制内部发色团的发射性质。mTFP0.7的时间分辨结构和光谱研究将揭示分子重排的细节，由于发色团异构化，这与许多生物分子（如参与视觉和光合作用的分子）的光物理学广泛相关，并将提供对其他类型荧光团观察到的瞬态光/暗切换过程的性质的洞察。从这项工作中获得的理解将是有用的，以改善这些和相关的荧光蛋白的性能，作为实用的，发光探针的活细胞的研究。预期用途的光活化探针，也被称为光学荧光笔，包括选择性标记的分子组装的研究细胞内的主动运输和发展的新技术，在高分辨率fluorescence microscopic.Broader ImpactsThe的跨学科性质的努力和各种各样的实验技术将提供优秀的多学科培训的学生和博士后研究人员。由于它们的视觉吸引力，易于生产和分离，加上特殊的物理稳定性，荧光蛋白为高中和大学的实验室课程提供了出色的教材，这项研究的结果将用于开发新的教学协议。最后，将通过互联网以教学录像和文字材料的形式向感兴趣的公众传播观察到的分子运动和光动力行为的其他细节的动画插图。

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 ImpactsThe 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.