Lanthanide-binding tags as luminescent probes for studying protein interactions.
J Am Chem Soc
; 128(22): 7346-52, 2006 Jun 07.
Article
en En
| MEDLINE
| ID: mdl-16734490
ABSTRACT
Herein, we report a method for studying protein-peptide interactions which exploits the luminescence properties of Tb(III). Lanthanide-binding tags (LBTs) are short peptide sequences comprising 15-20 naturally occurring amino acids that bind Tb(III) with high affinity. These genetically encodable luminescent tags are smaller in size than the Aequorea victoria fluorescent proteins (AFPs) and benefit from the long-lived luminescence lifetime of lanthanides. In this study, luminescence resonance energy transfer (LRET) was used to monitor the interaction between SH2 domains and different phosphopeptides. For the study, the SH2 domains of Src and Crk kinase were each coexpressed with an LBT, and phosphorylated and nonphosphorylated peptides were chemically synthesized with organic fluorophores. The LRET between the protein-bound Tb(III) and the peptide-based organic fluorophore was shown to be specific for the recognition of the SH2 domain and the peptide binding partner. This method can detect differences in binding affinity and can be used to measure the dissociation constant for the protein-peptide interaction. In addition, decay experiments can be used to calculate the distance between a site in the bound peptide and the protein using Förster theory. In all of these experiments, the millisecond luminescence lifetime of Tb(III) can be exploited using time-resolved detection to eliminate background fluorescence from organic fluorophores.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Péptidos
/
Terbio
/
Proteínas
/
Sustancias Luminiscentes
Idioma:
En
Revista:
J Am Chem Soc
Año:
2006
Tipo del documento:
Article
País de afiliación:
Estados Unidos