当前位置：
X-MOL 学术
›
Photochem. Photobiol.
›
论文详情
Our official English website, www.x-mol.net, welcomes your
feedback! (Note: you will need to create a separate account there.)
Elucidating Contributions from Multiple Species during Photoconversion of Enhanced Green Fluorescent Protein (EGFP) under Ultraviolet Illumination
Photochemistry and Photobiology ( IF 2.6 ) Pub Date : 2021-02-23 , DOI: 10.1111/php.13409 Shaina Dhamija 1 , Arijit K De 1
Photochemistry and Photobiology ( IF 2.6 ) Pub Date : 2021-02-23 , DOI: 10.1111/php.13409 Shaina Dhamija 1 , Arijit K De 1
Affiliation
|
Photocycle in wild-type green fluorescent protein (wt-GFP) involves generation of a bright fluorescent deprotonated chromophore from feebly fluorescent protonated form via excited-state proton transfer. In addition to this usual photocycle, wt-GFP is also known to exhibit irreversible photoconversion upon illumination with ultraviolet and visible radiation. However, a detailed understanding of photoconversion in enhanced GFP (EGFP: S65T/F64L mutant of wt-GFP), which predominantly exists in deprotonated form, is yet to be explored. Using 254 nm irradiation, we study how photoconversion proceeds in EGFP. The key findings are observation of spreading out of an isosbestic point and existence of an initial lag phase in spectral kinetics of absorbance, indicative of sequential photoconversion through an intermediate. Fluorescence kinetics of EGFP and its photoproduct are estimated by assigning two unique fluorescence lifetimes which is further complicated by the fact that their fluorescence are spectrally inseparable, as evident from global analysis of fluorescence lifetime. Time-resolved fluorescence anisotropy studies further suggest minimal structural changes in protein scaffold upon photoconversion. Based on these findings, an analytic model is developed to account for the overall decay in fluorescence (as photoconversion proceeds) that inherently incorporates the initial lag phase and a summary of energetics and processes involved is provided.
中文翻译：
在紫外线照射下增强型绿色荧光蛋白 (EGFP) 的光转换过程中阐明多种物种的贡献
野生型绿色荧光蛋白 ( wt -GFP) 中的光循环涉及通过激发态质子转移从微弱的荧光质子化形式产生明亮的荧光去质子化生色团。除了这种常见的光循环外，wt -GFP 还已知在紫外线和可见光照射下表现出不可逆的光转化。然而，详细了解增强型 GFP（EGFP： wt的 S65T/F64L 突变体）中的光转化-GFP)，主要以去质子化形式存在，尚待探索。使用 254 nm 照射，我们研究了 EGFP 中的光转换是如何进行的。主要发现是观察到等吸光点的扩散和吸收光谱动力学中存在初始滞后阶段，表明通过中间体的顺序光转换。EGFP 及其光产物的荧光动力学是通过指定两个独特的荧光寿命来估计的，这使它们的荧光在光谱上不可分割的事实进一步复杂化，这从荧光寿命的全局分析中可以看出。时间分辨荧光各向异性研究进一步表明光转换后蛋白质支架的结构变化很小。基于这些发现，
更新日期：2021-02-23
中文翻译：
在紫外线照射下增强型绿色荧光蛋白 (EGFP) 的光转换过程中阐明多种物种的贡献
野生型绿色荧光蛋白 ( wt -GFP) 中的光循环涉及通过激发态质子转移从微弱的荧光质子化形式产生明亮的荧光去质子化生色团。除了这种常见的光循环外，wt -GFP 还已知在紫外线和可见光照射下表现出不可逆的光转化。然而，详细了解增强型 GFP（EGFP： wt的 S65T/F64L 突变体）中的光转化-GFP)，主要以去质子化形式存在，尚待探索。使用 254 nm 照射，我们研究了 EGFP 中的光转换是如何进行的。主要发现是观察到等吸光点的扩散和吸收光谱动力学中存在初始滞后阶段，表明通过中间体的顺序光转换。EGFP 及其光产物的荧光动力学是通过指定两个独特的荧光寿命来估计的，这使它们的荧光在光谱上不可分割的事实进一步复杂化，这从荧光寿命的全局分析中可以看出。时间分辨荧光各向异性研究进一步表明光转换后蛋白质支架的结构变化很小。基于这些发现，
京公网安备 11010802027423号