Diffusion behavior of the fluorescent proteins eGFP and Dreiklang in solvents of different viscosity monitored by fluorescence correlation spectroscopy

Cornelia Junghans, F. Schmitt, V. Vukojević, T. Friedrich
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引用次数: 5

Abstract

Abstract Fluorescence correlation spectroscopy relies on temporal autocorrelation analysis of fluorescence intensity fluctuations that spontaneously arise in systems at equilibrium due to molecular motion and changes of state that cause changes in fluorescence, such as triplet state transition, photoisomerization and other photophysical transformations, to determine the rates of these processes. The stability of a fluorescent molecule against dark state conversion is of particular concern for chromophores intended to be used as reference tags for comparing diffusion processes on multiple time scales. In this work, we analyzed properties of two fluorescent proteins, the photoswitchable Dreiklang and its parental eGFP, in solvents of different viscosity to vary the diffusion time through the observation volume element by several orders of magnitude. In contrast to eGFP, Dreiklang undergoes a dark-state conversion on the time scale of tens to hundreds of microseconds under conditions of intense fluorescence excitation, which results in artificially shortened diffusion times if the diffusional motion through the observation volume is sufficiently slowed down. Such photophysical quenching processes have also been observed in FCS studies on other photoswitchable fluorescent proteins including Citrine, from which Dreiklang was derived by genetic engineering. This property readily explains the discrepancies observed previously between the diffusion times of eGFP- and Dreiklang-labeled plasma membrane protein complexes.
荧光相关光谱法监测了荧光蛋白eGFP和Dreiklang在不同黏度溶剂中的扩散行为
荧光相关光谱学依靠对平衡系统中由于分子运动和引起荧光变化的状态变化(如三重态跃迁、光异构化和其他光物理转化)而自发产生的荧光强度波动进行时间自相关分析,来确定这些过程的速率。荧光分子对暗态转换的稳定性是特别关注的发色团打算用作参考标签,比较在多个时间尺度上的扩散过程。在这项工作中,我们分析了两种荧光蛋白,光切换Dreiklang及其亲本eGFP,在不同粘度的溶剂中,通过观察体积元素改变扩散时间的几个数量级。与eGFP不同的是,在强烈的荧光激发条件下,Dreiklang在几十到几百微秒的时间尺度上发生暗态转换,如果充分减慢扩散运动在观察体积中的速度,就会人为地缩短扩散时间。这种光物理猝灭过程也在其他可光切换荧光蛋白的FCS研究中被观察到,包括通过基因工程获得Dreiklang的Citrine。这一特性很容易解释先前观察到的eGFP-和dreiklang标记的质膜蛋白复合物在扩散时间上的差异。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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