Quantitative Determination of Dark Chromophore Population Explains the Apparent Low Quantum Yield of Red Fluorescent Proteins
| Autor: | Laura van Weeren, Christian Blum, Jente Stouthamer, Daphne S. Bindels, Vinod Subramaniam, Theodorus W. J. Gadella, Jord C. Prangsma, Robert Molenaar, Lindsay Haarbosch, Willem L. Vos |
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| Přispěvatelé: | Molecular Cytology (SILS, FNWI), Nanobiophysics, Complex Photonic Systems |
| Rok vydání: | 2020 |
| Předmět: |
genetic structures
Population UT-Hybrid-D Quantum yield Astrophysics::Cosmology and Extragalactic Astrophysics 02 engineering and technology Molecular physics Article Fluorescence 03 medical and health sciences Materials Chemistry Particle Size Physical and Theoretical Chemistry education Quantum 030304 developmental biology Physics 0303 health sciences education.field_of_study Chromophore 021001 nanoscience & nanotechnology Surfaces Coatings and Films Luminescent Proteins Förster resonance energy transfer Microscopy Fluorescence Excited state sense organs 0210 nano-technology mCherry |
| Zdroj: | The journal of Physical Chemistry. B, 124(8), 1383-1391. American Chemical Society The Journal of physical chemistry B, 124(8), 1383-1391. American Chemical Society The Journal of Physical Chemistry. B |
| ISSN: | 1520-5207 1520-6106 |
| Popis: | The fluorescence quantum yield of four representative red fluorescent proteins mCherry, mKate2, mRuby2, and the recently introduced mScarlet was investigated. The excited state lifetimes were measured as a function of the distance to a gold mirror in order to control the local density of optical states (LDOS). By analyzing the total emission rates as a function of the LDOS, we obtain separately the emission rate and the nonradiative rate of the bright states. We thus obtain for the first time the bright state quantum yield of the proteins without interference from dark, nonemitting states. The bright state quantum yields are considerably higher than previously reported quantum yields that average over both bright and dark states. We determine that mCherry, mKate2, and mRuby2 have a considerable fraction of dark chromophores up to 45%, which explains both the low measured quantum yields of red emitting proteins reported in the literature and the difficulties in developing high quantum yield variants of such proteins. For the recently developed bright mScarlet, we find a much smaller dark fraction of 14%, accompanied by a very high quantum yield of the bright state of 81%. The presence of a considerable fraction of dark chromophores has implications for numerous applications of fluorescent proteins, ranging from quantitative fluorescence microscopy to FRET studies to monitoring protein expression levels. We recommend that future optimization of red fluorescent proteins should pay more attention to minimizing the fraction of dark proteins. |
| Databáze: | OpenAIRE |
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