Photodynamics of Fluorescent Proteins

MTFP0.7是一种明亮的单体青色荧光蛋白，但与红礁和黄礁GFP具有很高的序列和结构同源性。静态和时间分辨X射线结晶学、超快光谱和突变分析将被用来研究mTFP 0.7显著的光动力学行为。这种蛋白质在荧光状态和非荧光状态之间进行简单的光切换，而衍生的但密切相关的蛋白质mTPF1是光稳定性的。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特性。MTFP0.7的时间分辨结构和光谱研究将揭示由于生色团异构化而导致的分子重排的细节，这些细节与许多生物分子(如涉及视觉和光合作用的分子)的光物理相关，并将提供对其他类型荧光团观察到的瞬时光/暗切换过程的本质的洞察。从这项工作中获得的理解将有助于改进这些和相关荧光蛋白的性质，将其用作活细胞研究的实用发光探针。可光激活探针的预期用途，也被称为光学荧光笔，包括选择性标记分子组件以研究细胞内的活性运输，以及开发高分辨率荧光显微镜的新技术。广泛影响这一努力的跨学科性质和种类繁多的实验技术将为学生和博士后研究人员提供出色的多学科培训。由于它们的视觉吸引力、易于生产和分离以及特殊的物理稳定性，荧光蛋白质为高中和大学的实验室课程提供了优秀的教材，这项研究的结果将用于开发新的教学方案。最后，观察到的分子运动的动画插图和光动力学行为的其他细节将以教学视频和文本材料的形式通过互联网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.