TIME-RESOLVED STRUCTURAL STUDIES OF MTFP07 FLUORESCENCE PHOTOSWITCHING

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们建议继续在mTFP 0.7荧光光开关的基础上进行结构研究，mTFP 0.7是一种与绿色荧光蛋白(GFP)有远缘关系的单体青色荧光蛋白。MTFP0.7最初是明亮的荧光，峰值发射波长为488 nm，然而，当蛋白质被蓝光(~450 nm)照射时，会迅速转换为非荧光形式[1]。在室温下，观察到自发荧光恢复到接近100%的时间尺度约为8分钟。光开关的mTFP0.7可以通过360-400 nm的光曝光而非常迅速地重新激活。MTFP0.7的结构已经在荧光和低温捕获的非荧光状态下得到了解决[2]。这表明了排放特性变化的一种机制。在荧光状态下，发色团是顺式共面的(就像在绝大多数明亮的荧光蛋白中发现的那样)，而在暗非荧光状态下，发色团是反式的、高度扭曲的、质子化的和部分无序的。我们的结论是，后三个特征都可以被认为是较低的发射效率，综合起来为荧光的完全丧失提供了一个令人信服的解释。这些特性为光开关提供了一个有用的模型，适用于迄今为止所描述的所有可光开关的荧光蛋白。此外，内部侧链的戏剧性重新排列意味着一把锁和钥匙。解释非荧光状态长寿命的机制。对mTFP0.7的时间分辨结晶学研究试图揭示光开关现象的基础。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We propose continued structural studies on the basis of fluorescence photoswitching in mTFP0.7, a monomeric cyan fluorescent protein distantly related to the green fluorescent protein (GFP). mTFP0.7 is initially brightly fluorescent with a peak emission wavelength of 488 nm, however, rapid photoswitching to a nonfluorescent form occurs when the protein is illuminated with blue light (~ 450 nm)[1]. Spontaneous fluorescence recovery to nearly 100% is observed on a time scale of about 8 minutes at room temperature. Photoswitched mTFP0.7 can be very rapidly reactivated by exposure to 360-400 nm light. Structures of mTFP0.7 have been solved in the fluorescent and cryo-trapped nonfluorescent states[2]. These suggest a mechanism for the change in emission characteristics. In the fluorescent state, the chromophore is cis-coplanar (as found in the vast majority of brightly fluorescent proteins), however, in the dark nonfluorescent state, the chromophore is trans, highly twisted, protonated and partially disordered. We concluded that the latter three characteristics could each be argued to lower emission efficiency and taken together provide a compelling explanation for the complete loss of fluorescence. These characteristics provide a useful model for photoswitching, applicable to all photoswitchable fluorescent proteins described to date. Furthermore, a dramatic rearrangement of internal side chains suggests a ?lock and key? mechanism to explain the long life of the nonfluorescent state. The proposed time-resolved crystallographic studies on mTFP0.7 seek to reveal the basis for the photoswitching phenomenon.