在单分子FRET实验中的光漂白途径
Xiangxu Kong1, Eyal Nir, Kambiz Hamadani
1Department of Chemistry and Biochemistry, and California NanoSystems Institute, University of California-Los Angeles, Los Angeles, CA 90095, USA.
Journal of the American Chemical Society
|March 23, 2007
概括
了解单分子光共振能量转移 (sm-FRET) 中的受体光漂白至关重要. 我们发现接受器光漂白与FRET效率和供体激发有关,这表明脉冲供体激发可以改善sm-FRET实验.
科学领域:
- 生物物理学的生物物理.
- 频谱学是一种光谱学.
- 分子机器分子机器
背景情况:
- 单分子光共振能量转移 (sm-FRET) 需要高光子流量来进行准确的生物分子机器分析.
- 高激光激发强度,在增加光子流量的同时,加速光漂白,限制实验持续时间.
- 抗氧化剂添加剂通常用于减轻受体光漂白.
研究的目的:
- 为了阐明sm-FRET中受体的主要光漂白路径.
- 确定影响受体光漂白率的因素.
- 在sm-FRET测量中提出减少接受器光漂白的策略.
主要方法:
- 利用先进的单分子和合体光谱技术.
- 采用双重标记的Acyl-CoA结合蛋白和双链DNA作为模型系统.
- 在不同的激发条件和FRET效率下研究光漂白途径.
主要成果:
- 接收器光漂白率与FRET效率呈正相关性.
- 与连续波激发相比,皮秒脉冲激发增强了接受器光漂白.
- 接受器光漂白强度尺度与捐赠者激发激光强度,而不是接受器激光强度.
结论:
- 主要的受体光漂白途径涉及从受体的第一个激发单元状态吸收短波长光子.
- 捐赠者的光漂白通常不是sm-FRET的限制因素.
- 采用脉冲供体激发是一种潜在的策略,以尽量减少sm-FRET中的接受器光漂白.
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