Quantification of photosensitized singlet oxygen production by a fluorescent protein.
Quantification of photosensitized singlet oxygen production by a fluorescent protein.
复制标题
DOI：
10.1002/cphc.201000919
复制
发表时间：
2011-01
期刊：
影响因子：
--
通讯作者：
X. Ragàs;L. P. Cooper;John H. White;S. Nonell;C. Flors
中科院分区：
文献类型：
--
作者：
X. Ragàs;L. P. Cooper;John H. White;S. Nonell;C. Flors
荧光蛋白越来越多地成为一系列细胞生物学技术中的致动器。这些技术之一是发色团辅助激光灭活（CALI），其用于通过产生光化学损伤来特异性地破坏靶蛋白或细胞器的功能。CALI通过照射能够产生活性氧（ROS）的染料来实现。CALI和荧光蛋白提供的标记特异性的组合对于避免不受控制的光损伤是有用的，尽管ROS的失活机制依赖于荧光蛋白并且尚未完全理解。在此，我们提出了一个定量研究的能力的红色荧光蛋白TagRFP产生活性氧，特别是单线态氧（（1）O（2））。TagRFP能够光敏化（1）O（2），估计量子产率为0.004。这是对GFP样蛋白质的（1）O（2）生产值的量子产率的首次估计。我们还发现，TagRFP有一个短的三重态寿命相比，绿色荧光蛋白，这反映了相对较高的氧气可及性的发色团。对TagRFP的结构和生物物理性质的洞察在改进用于荧光显微镜和CALI的荧光蛋白方面具有意义。
Fluorescent proteins are increasingly becoming actuators in a range of cell biology techniques. One of those techniques is chromophore-assisted laser inactivation (CALI), which is employed to specifically inactivate the function of target proteins or organelles by producing photochemical damage. CALI is achieved by the irradiation of dyes that are able to produce reactive oxygen species (ROS). The combination of CALI and the labelling specificity that fluorescent proteins provide is useful to avoid uncontrolled photodamage, although the inactivation mechanisms by ROS are dependent on the fluorescent protein and are not fully understood. Herein, we present a quantitative study of the ability of the red fluorescent protein TagRFP to produce ROS, in particular singlet oxygen ((1)O(2)). TagRFP is able to photosensitize (1)O(2) with an estimated quantum yield of 0.004. This is the first estimation of a quantum yield of (1)O(2) production value for a GFP-like protein. We also find that TagRFP has a short triplet lifetime compared to EGFP, which reflects relatively high oxygen accessibility to the chromophore. The insight into the structural and photophysical properties of TagRFP has implications in improving fluorescent proteins for fluorescence microscopy and CALI.