Characterization of Fluorescein Arsenical Hairpin (FlAsH) as a Probe for Single-Molecule Fluorescence Spectroscopy.
Sci Rep
; 7(1): 13063, 2017 10 12.
Article
en En
| MEDLINE
| ID: mdl-29026195
ABSTRACT
In recent years, new labelling strategies have been developed that involve the genetic insertion of small amino-acid sequences for specific attachment of small organic fluorophores. Here, we focus on the tetracysteine FCM motif (FLNCCPGCCMEP), which binds to fluorescein arsenical hairpin (FlAsH), and the ybbR motif (TVLDSLEFIASKLA) which binds fluorophores conjugated to Coenzyme A (CoA) via a phosphoryl transfer reaction. We designed a peptide containing both motifs for orthogonal labelling with FlAsH and Alexa647 (AF647). Molecular dynamics simulations showed that both motifs remain solvent-accessible for labelling reactions. Fluorescence spectra, correlation spectroscopy and anisotropy decay were used to characterize labelling and to obtain photophysical parameters of free and peptide-bound FlAsH. The data demonstrates that FlAsH is a viable probe for single-molecule studies. Single-molecule imaging confirmed dual labeling of the peptide with FlAsH and AF647. Multiparameter single-molecule Förster Resonance Energy Transfer (smFRET) measurements were performed on freely diffusing peptides in solution. The smFRET histogram showed different peaks corresponding to different backbone and dye orientations, in agreement with the molecular dynamics simulations. The tandem of fluorophores and the labelling strategy described here are a promising alternative to bulky fusion fluorescent proteins for smFRET and single-molecule tracking studies of membrane proteins.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Arsenicales
/
Espectrometría de Fluorescencia
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Imagen Individual de Molécula
Idioma:
En
Revista:
Sci Rep
Año:
2017
Tipo del documento:
Article
País de afiliación:
Canadá
Pais de publicación:
ENGLAND
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ESCOCIA
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GB
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GREAT BRITAIN
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INGLATERRA
/
REINO UNIDO
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SCOTLAND
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UK
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UNITED KINGDOM