We study the fluorescence quantum efficiency of four representative red fluorescent proteins mCherry, mKate2, mRuby2 and the recently introduced mScarlet. We measure the excited state lifetimes as a function of the distance to a gold mirror in order to control the local density of states (LDOS). By analyzing the total emission rates as a function of the LDOS, we obtain separately the emission rate of the bright states and the non-radiative rate of all molecules. We thus obtain for the first time ever the bright state quantum efficiency of the proteins without influence from dark, non-emitting states. The bright state quantum efficiencies are remarkably higher than previously reported efficiencies that average both bright and dark states. We deduce that mCherry, mKate2 and mRuby2 have a considerable fraction of dark molecules up to more than 40%, which explains both the low quantum efficiencies of red emitting proteins reported in literature and the difficulties in developing high quantum efficiency variants of such proteins. For the recently developed bright mScarlet we find a much smaller dark fraction of only 14 %. The presence of a considerable fraction of dark chromophores has implications for numerous applications of fluorescent proteins, ranging from FRET studies, and quantitative fluorescence microscopy to monitoring protein expression levels. We recommend that future optimization of red fluorescent proteins should concentrate on minimizing the fraction of dark proteins.

https://dx.doi.org/10.1021/acs.jpcb.9b1039

J. Prangsma, R. Molenaar, L. van Weeren, D. Bindels, L. Haarbosch, J. Stouthamer, T. Gadella, V. Subramaniam, W. L. Vos, and C. Blum

2020

02/2020

J. Phys. Chem. B

124

1383−1391

10.1021/acs.jpcb.9b1039