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荧光蛋白具有高度的序列和结构同源性。静态和时间分辨x射线晶体学、超快光谱和突变分析将用于研究mTFP0.7的显著光动力学行为。该蛋白在荧光和非荧光状态之间进行容易的光转换，而衍生的密切相关的蛋白mTPF1是光稳定的。光谱学、热力学和比较结构研究将有助于了解蛋白质的三维结构如何控制内部发色团的发射特性。mTFP0.7的时间分辨结构和光谱研究将揭示由于发色团异构化而导致的分子重排的细节，这与许多生物分子（例如涉及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见解。从这项工作中获得的理解将有助于改善这些和相关荧光蛋白的特性，将其用作研究活细胞的实用发光探针。光活化探针的预期用途，也被称为光学荧光笔，包括选择性标记分子组装，用于研究细胞内的主动运输，以及开发高分辨率荧光显微镜的新技术。更广泛的影响这项工作的跨学科性质和各种各样的实验技术将为学生和博士后研究人员提供优秀的多学科培训。由于荧光蛋白具有视觉吸引力，易于生产和分离，并且具有特殊的物理稳定性，因此荧光蛋白为高中和大学的实验课程提供了出色的教学材料，本研究的结果将用于开发新的教学方案。最后，观察到的分子运动和其他光动力学行为细节的动画插图将通过互联网以教学视频和文本材料的形式传播给感兴趣的公众。

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.