Characterization of Fluorescent Proteins with Intramolecular Photostabilization*.
Chembiochem
; 22(23): 3283-3291, 2021 12 02.
Article
em En
| MEDLINE
| ID: mdl-34296494
ABSTRACT
Genetically encodable fluorescent proteins have revolutionized biological imaging inâ
vivo and inâ
vitro. Despite their importance, their photophysical properties, i. e., brightness, count-rate and photostability, are relatively poor compared to synthetic organic fluorophores or quantum dots. Intramolecular photostabilizers were recently rediscovered as an effective approach to improve photophysical properties of organic fluorophores. Here, direct conjugation of triplet-state quenchers or redox-active substances creates high local concentrations of photostabilizer around the fluorophore. In this paper, we screen for effects of covalently linked photostabilizers on fluorescent proteins. We produced a double cysteine mutant (A206C/L221C) of α-GFP for attachment of photostabilizer-maleimides on the ß-barrel near the chromophore. Whereas labelling with photostabilizers such as trolox, aâ
nitrophenyl group, and cyclooctatetraene, which are often used for organic fluorophores, had no effect on α-GFP-photostability, a substantial increase of photostability was found upon conjugation to azobenzene. Although the mechanism of the photostabilizing effects remains to be elucidated, we speculate that the higher triplet-energy of azobenzene might be crucial for triplet-quenching of fluorophores in the blue spectral range. Our study paves the way for the development of fluorescent proteins with photostabilizers in the protein barrel by methods such as unnatural amino acid incorporation.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fármacos Fotossensibilizantes
/
Proteínas de Fluorescência Verde
/
Proteínas Luminescentes
Idioma:
En
Revista:
Chembiochem
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Holanda