|EC (M-1 cm-1)
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This strongly suggests that the fluorescent protein tag caused clustering artifacts (Fig. 2a) and that ClpX and ClpP fluorescent protein fusions cannot be trusted for determining localization of native, untagged proteins. [...] These findings motivated us to evaluate other fluorescent proteins fused to ClpP or ClpX. We found that sfGFP, Venus, mCherry and mCherry2 all caused substantial foci formation, despite being monomers or very weak dimers when expressed alone. mKate2 and TagRFP-T caused intermediate clustering, whereas with mVenus and mYPet most of the fluorescence signal was spatially uniform, although we observed foci in a few cells. mTagBFP and mEos2 fusions resulted in a weak signal with infrequent dim foci. We detected no foci for photoswitchable (PS)-CFP2, reversible switchable (rs)FastLime (data not shown) and a mutant of sfGFP with a charge of −30 (GFP(−30)), but the signals were very dim. Finally, mGFPmut3, Dronpa and Dendra2 displayed an essentially uniform signal. Fluorescent protein fusions to ClpP generally caused more foci formation than fusions to ClpX, in particular for mYPet (Fig. 2d).
mFruit variant mCherry2 is a further engineered variant of mCherry that retains similar excitation and emission maxima (λex = 589 nm and λem = 610 nm) but has slightly higher brightness. This variant was generated some years ago by several rounds of directed evolution of an mCherry-ferritin fusion, as was originally described for Superfolder GFP (Waldo et al, 1999), but was not characterized in detail until the current work
The increased in vitro brightness of mCherry2 does not translate into substantially improved brightness in either bacteria or mammalian cells. However, we noticed that E. coli colonies expressing mCherry2 have a larger size than the ones expressing mCherry, at similar colony densities on LB agar plates. This observation suggested that mCherry2 might have decreased cytotoxicity in E. coli.
mCherry2, a not fully characterized mCherry variant, possesses superior properties compared to all other tested red FPs, i.e. mCherry, mCardinal, mRuby3, mScarlet and mScarlet-I. Additionally, by performing Fluorescence Cross-Correlation Spectroscopy (FCCS) measurements of FP hetero-dimers, we show that mCherry2 improves the quantification of the spectral cross-correlation compared to mCherry and propose to use mEGFP and mCherry2 as a novel standard FP pair for hetero-interaction studies.