Improved Chemical-Genetic Fluorescent Markers for Live Cell Microscopy.
Biochemistry
; 57(39): 5648-5653, 2018 10 02.
Article
em En
| MEDLINE
| ID: mdl-30204425
ABSTRACT
Inducible chemical-genetic fluorescent markers are promising tools for live cell imaging requiring high spatiotemporal resolution and low background fluorescence. The fluorescence-activating and absorption shifting tag (FAST) was recently developed to form fluorescent molecular complexes with a family of small, synthetic fluorogenic chromophores (so-called fluorogens). Here, we use rational design to modify the binding pocket of the protein and screen for improved fluorescence performances with four different fluorogens. The introduction of a single mutation results in improvements in both quantum yield and dissociation constant with nearly all fluorogens tested. Our improved FAST (iFAST) allowed the generation of a tandem iFAST (td-iFAST) that forms green and red fluorescent reporters 1.6-fold and 2-fold brighter than EGFP and mCherry, respectively, while having a comparable size.
Texto completo:
1
Coleções:
01-internacional
Contexto em Saúde:
3_ND
Base de dados:
MEDLINE
Assunto principal:
Rodanina
/
Proteínas de Bactérias
/
Fotorreceptores Microbianos
/
Corantes Fluorescentes
Limite:
Humans
Idioma:
En
Revista:
Biochemistry
Ano de publicação:
2018
Tipo de documento:
Article