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mScarlet: a bright monomeric red uorescent protein for cellular imaging
Daphne S Bindels1,4, Lindsay Haarbosch1,4,
Laura van Weeren1, Marten Postma1, Katrin E Wiese1, Marieke Mastop1, Sylvain Aumonier2,3,
Guillaume Gotthard2,3, Antoine Royant2,3,
Mark A Hink1 & Theodorus W J Gadella Jr1
We report the engineering of mScarlet, a truly monomeric red uorescent protein with record brightness, quantum yield (70%) and uorescence lifetime (3.9 ns). We developed mScarlet starting with a consensus synthetic template and using improved spectroscopic screening techniques; mScarlets crystal structure reveals a planar and rigidied chromophore. mScarlet outperforms existing red uorescent proteins as a fusion tag, and it is especially useful as a Frster resonance energy transfer (FRET) acceptor in ratiometric imaging.
Fluorescent proteins (FPs) have become indispensable in biological research1. After the cloning of GFP from the jellyfish Aequorea victoria2, several GFP spectral variants were developed, including blue, cyan and yellow FPs3. The palette of FPs was greatly expanded after the cloning of red FP (RFP) homologs from corals and other Anthozoa species4,5. However, all Anthozoa RFPs form obligate tetramers, which can seriously interfere with localization and functioning of RFP-fusion proteins. Monomerization of tetrameric RFPs was accompanied by a serious deterioration of the brightness and incomplete and/or partial green maturation of the resulting monomer6. After the development of the first monomeric RFP (mRFP), mRFP1 (ref. 6), several improved mRFPs have been reported: mCherry7, mApple8, TagRFP(-T)8,9, mKate2 (ref. 10), mRuby2 (ref. 11), mRuby3 (ref. 12) and FusionRed13 (reviewed in ref. 14). But all these mRFPs are dimmer than their tetrameric ancestors; they have quantum yields below 50%, and several still harbor additional problems due to incomplete or partial green maturation and a residual tendency to dimerize15.
Spectral variants of FPs can be applied in FRET-based biosensors to probe molecular interactions, conformational changes and metabolite concentrations within living cells16. While good FP-based FRET pairs are available with cyan FPs (CFPs) as donors
1Section of Molecular Cytology and van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, the Netherlands. 2European Synchrotron Radiation Facility, Grenoble, France. 3Institut de Biologie Structurale, Universit Grenoble Alpes, CNRS, CEA, Grenoble, F-38044, France. 4These authors contributed equally to this work. Correspondence should be addressed to T.W.J.G. ([email protected]).
RECEIVED 29 JULY; ACCEPTED 20 OCTOBER; PUBLISHED...