文章基本信息

标题：Optimal fluorescent protein tags for quantifying protein oligomerization in living cells
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作者：Valentin Dunsing ; Madlen Luckner ; Boris Zühlke 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2018
卷号：8
期号：1
页码：10634
DOI：10.1038/s41598-018-28858-0
语种：English
出版社：Springer Nature
摘要：Fluorescence fluctuation spectroscopy has become a popular toolbox for non-disruptive analysis of molecular interactions in living cells. The quantification of protein oligomerization in the native cellular environment is highly relevant for a detailed understanding of complex biological processes. An important parameter in this context is the molecular brightness, which serves as a direct measure of oligomerization and can be easily extracted from temporal or spatial fluorescence fluctuations. However, fluorescent proteins (FPs) typically used in such studies suffer from complex photophysical transitions and limited maturation, inducing non-fluorescent states. Here, we show how these processes strongly affect molecular brightness measurements. We perform a systematic characterization of non-fluorescent states for commonly used FPs and provide a simple guideline for accurate, unbiased oligomerization measurements in living cells. Further, we focus on novel red FPs and demonstrate that mCherry2, an mCherry variant, possesses superior properties with regards to precise quantification of oligomerization.