TIME-RESOLVED STRUCTURAL STUDIES OF MTFP07 FLUORESCENCE PHOTOSWITCHING

MTFP07 荧光光开关的时间分辨结构研究

• 批准号: 8171976
• 负责人: S. James REMINGTON
• 金额: $ 1.46万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171976
• 关键词: Characteristics; Computer Retrieval of Information on Scientific Projects Database; Cyan Fluorescent Protein; DNA Sequence Rearrangement; Disease; Exposure to; Fluorescence; Funding; Grant; Green Fluorescent Proteins; Institution; Life; Light; Modeling; Proteins; Recovery; Research; Research Personnel; Resources; Side; Source; Structure; Temperature; Time; United States National Institutes of Health; base; chromophore

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.

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们建议继续结构研究的基础上的荧光光开关在mTFP0.7，单体青色荧光蛋白远亲的绿色荧光蛋白（GFP）。mTFP0.7最初是明亮的荧光，峰值发射波长为488 nm，然而，当用蓝光（~ 450 nm）照射蛋白质时，会发生快速光转换为非荧光形式[1]。在室温下，在约8分钟的时间尺度上观察到自发荧光恢复到接近100%。光开关mTFP0.7可以通过暴露于360-400 nm光而非常迅速地重新激活。mTFP0.7的结构已在荧光和低温捕获的非荧光状态下得到解决[2]。这表明了排放特性变化的机制。在荧光状态下，发色团是顺式共面的（如在绝大多数亮荧光蛋白中发现的），然而，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，后三个特征可以被认为是降低发射效率，并结合在一起提供了一个令人信服的解释完全丧失荧光。这些特性提供了一个有用的模型，适用于所有的光开关荧光蛋白描述的日期。此外，一个戏剧性的内部侧链重排表明？锁和钥匙？机制来解释非荧光状态的长寿命。提出的时间分辨晶体学研究mTFP0.7试图揭示光开关现象的基础。