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Zoekresultaten

Zoekopdracht: faculteit: "FNWI" en publicatiejaar: "2012"

AuteursJ. Goedhart, D. von Stetten, M. Noirclerc-Savoye, M. Lelimousin, L. Joosen, M.A. Hink, L. van Weeren, Th.W.J. Gadella, A. Royant
TitelStructure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%.
TijdschriftNature Communications
Jaargang3
Jaar2012
Pagina's-751
ISSN20411723
FaculteitFaculteit der Natuurwetenschappen, Wiskunde en Informatica
Instituut/afd.FNWI: Swammerdam Institute for Life Sciences (SILS)
SamenvattingCyan variants of green fluorescent protein are widely used as donors in Forster resonance energy transfer experiments. The popular, but modestly bright, Enhanced Cyan Fluorescent Protein (ECFP) was sequentially improved into the brighter variants Super Cyan Fluorescent Protein 3A (SCFP3A) and mTurquoise, the latter exhibiting a high-fluorescence quantum yield and a long mono-exponential fluorescence lifetime. Here we combine X-ray crystallography and excited-state calculations to rationalize these stepwise improvements. The enhancement originates from stabilization of the seventh beta-strand and the strengthening of the sole chromophore-stabilizing hydrogen bond. The structural analysis highlighted one suboptimal internal residue, which was subjected to saturation mutagenesis combined with fluorescence lifetime-based screening. This resulted in mTurquoise2, a brighter variant with faster maturation, high photostability, longer mono-exponential lifetime and the highest quantum yield measured for a monomeric fluorescent protein. Together, these properties make mTurquoise2 the preferable cyan variant of green fluorescent protein for long-term imaging and as donor for Forster resonance energy transfer to a yellow fluorescent protein.
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