mScarlet3: a brilliant and fast-maturing red fluorescent protein

We report the evolution of mScarlet3, a cysteine-free monomeric red fluorescent protein with fast and complete maturation, as well as record brightness, quantum yield (75%) and fluorescence lifetime (4.0 ns). The mScarlet3 crystal structure reveals a barrel rigidified at one of its heads by a large hydrophobic patch of internal residues. mScarlet3 behaves well as a fusion tag, displays no apparent cytotoxicity and it surpasses existing red fluorescent proteins as a Forster resonance energy transfer acceptor and as a reporter in transient expression systems. Two mScarlet variants with high brightness and fast maturation times have been evolved. These variants behave favorably as fusion tags and Forster resonance energy transfer acceptors.

Automated summary

We have evolved mScarlet3, a cysteine-free monomeric red fluorescent protein, with fast and complete maturation, high brightness, quantum yield (75%) and fluorescence lifetime (4.0 ns). The crystal structure of mScarlet3 reveals a barrel rigidified at one of its heads by a large hydrophobic patch of internal residues. mScarlet3 performs well as a fusion tag, shows no apparent cytotoxicity, and outperforms existing red fluorescent proteins as a Forster resonance energy transfer acceptor and as a reporter in transient expression systems.