Minimum set of mutations needed to optimize cyan fluorescent proteins for live cell imaging - Biological Adaptation and Ageing Accéder directement au contenu
Article Dans Une Revue Molecular BioSystems Année : 2013

Minimum set of mutations needed to optimize cyan fluorescent proteins for live cell imaging

Marie Erard
Asma Fredj
  • Fonction : Auteur
Helene Pasquier
  • Fonction : Auteur
Dahdjim-Benoit Beltolngar
  • Fonction : Auteur
Yasmina Bousmah
  • Fonction : Auteur
Valerie Derrien
Fabienne Merola
  • Fonction : Auteur
  • PersonId : 841250

Résumé

Cyan fluorescent proteins (CFPs) are widely used as FRET donors in genetically encoded biosensors for live cell imaging. Recently, cyan variants with greatly improved fluorescence quantum yields have been developed by large scale random mutagenesis. We show that the introduction of only two mutations, T65S and H148G, is able to confer equivalent performances on the popular form ECFP, leading to Aquamarine (QY = 0.89, tau(f) = 4.12 ns). Besides an impressive pH stability (pK(1/2) = 3.3), Aquamarine shows a very low general sensitivity to its environment, and undetectable photoswitching reactions. Aquamarine gives efficient and bright expression in different mammalian cell systems, with a long and single exponential intracellular fluorescence lifetime mostly insensitive to the fusion or the subcellular location of the protein. Aquamarine was also able to advantageously replace the CFP donor in the FRET biosensor AKAR for ratiometric measurements of protein kinase A activity. The performances of Aquamarine show that only two rounds of straightforward single point mutagenesis can be a quick and efficient way to optimize the donor properties in FRET-based biosensors.
Fichier non déposé

Dates et versions

hal-01545402 , version 1 (22-06-2017)

Identifiants

Citer

Marie Erard, Asma Fredj, Helene Pasquier, Dahdjim-Benoit Beltolngar, Yasmina Bousmah, et al.. Minimum set of mutations needed to optimize cyan fluorescent proteins for live cell imaging. Molecular BioSystems, 2013, 9 (2), pp.258-267. ⟨10.1039/c2mb25303h⟩. ⟨hal-01545402⟩
87 Consultations
0 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More