Quantifying protein oligomerization in living cells: A systematic comparison of fluorescent proteins

摘要: Fluorescence fluctuation spectroscopy has become a popular toolbox for non-disruptive studies of molecular interactions and dynamics in living cells. The quantification e.g. protein oligomerization absolute concentrations the native cellular environment is highly relevant detailed understanding complex signaling pathways biochemical reaction networks. A parameter particular relevance this context brightness, which serves as direct measure can be easily extracted from temporal or spatial fluorescence fluctuations. However, fluorescent proteins (FPs) typically used such suffer photophysical transitions limited maturation, potentially inducing non-fluorescent states, strongly affect brightness measurements. Although these processes have been occasionally reported, comprehensive study addressing issue missing. Here, we investigate suitability commonly FPs (i.e. mEGFP, mEYFP mCherry), well novel red mCherry2, mRuby3, mCardinal, mScarlet mScarlet-I) based on obtained by Correlation Spectroscopy (FCS) Number&Brightness (N&B) measurements For all FPs, measured lower than expected FP homo-dimers, allowing us to estimate, each label, probability emission simple two-state model. By analyzing higher homo-oligomers Influenza virus Hemagglutinin (HA) protein, show that oligomeric state complexes only accurately quantified if taken into account. Further, provide strong evidence an mCherry variant, possesses superior apparent probability, presumably due its fast maturation. We finally conclude property leads improved cross-correlation propose use mEGFP mCherry2 standard pair studying biomolecular hetero-interactions.