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

Abstract

Fluorescence fluctuation spectroscopy has become a popular toolbox for non-disruptive studies of molecular interactions and dynamics in living cells. The quantification of e.g. protein oligomerization and absolute concentrations in the native cellular environment is highly relevant for a detailed understanding of complex signaling pathways and biochemical reaction networks. A parameter of particular relevance 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, potentially inducing non-fluorescent states, which strongly affect molecular brightness measurements. Although these processes have been occasionally reported, a comprehensive study addressing this issue is missing. Here, we investigate the suitability of commonly used FPs (i.e. mEGFP, mEYFP and mCherry), as well as novel red FPs (i.e. mCherry2, mRuby3, mCardinal, mScarlet and mScarlet-I) for the quantification of oligomerization based on the molecular brightness, as obtained by Fluorescence Correlation Spectroscopy (FCS) and Number&Brightness (N&B) measurements in living cells. For all FPs, we measured a lower than expected brightness of FP homo-dimers, allowing us to estimate, for each fluorescent label, the probability of fluorescence emission in a simple two-state model. By analyzing higher FP homo-oligomers and the Influenza A virus Hemagglutinin (HA) protein, we show that the oligomeric state of protein complexes can only be accurately quantified if this probability is taken into account. Further, we provide strong evidence that mCherry2, an mCherry variant, possesses a superior apparent fluorescence probability, presumably due to its fast maturation. We finally conclude that this property leads to an improved quantification in fluorescence cross-correlation spectroscopy measurements and propose to use mEGFP and mCherry2 as the novel standard pair for studying biomolecular hetero-interactions. * Measured at four times higher laser power (19.6 µW) than used in N&B measurements ** Average molecular brightness in N&B, interpolated to the same laser power (4.9 µW) for all red FPs

Multi-color fluorescence fluctuation spectroscopy in living cells via spectral detection

“…This value corresponds to a pf of ca. 70% for mEGFP, as expected (14). To confirm that absolute brightness values are not influenced by the spectral decomposition, we also determined the brightness of mEGFP in cells expressing mp-mEGFP alone ( Fig.3G), resulting in values close to 1 (1.08±0.23, mean±SD, n=28 cells).…”

“…Data acquisition: RSICS measurements were performed as previously described (61) In these cases, all pixels were selected and minor brightness differences between cytoplasm and nucleus, previously found to be ca. 10% (14), were neglected. Image stacks were further processed with a high-pass filter (with a moving 4-frame window) to remove slow signal − 1 (14).…”

“…10% (14), were neglected. Image stacks were further processed with a high-pass filter (with a moving 4-frame window) to remove slow signal − 1 (14).…”

“…The molecular brightness was calculated by dividing the mean count rate detected for each species i by the particle number Ni determined from the fit: < = 〈V W (X)〉 q W . From this value, an estimate of the oligomeric state < was determined by normalizing Bi by the average molecular brightness Bi,1 of the corresponding monomeric reference, and, subsequently, by the fluorescence probability pf,i for species i: < = / W // W,r s t,W − 1, as previously derived (14). The pf was previously characterized for several FPs, e.g.…”

“…Afterwards, RICS spatial ACFs and pair-wise CCFs were calculated for each image stack and all combinations of species i, j (e.g. G and Y, G and Ch2, Y and Ch2 for three species), respectively(27,35): the fluorescence probability pf,i for species i: = / ,as previously derived(14). The pf was calculated from the obtained molecular brightness Bi,2 of FP homodimers of species i:…”