Simple and efficient strategy for site-specific dual labeling of proteins for single-molecule fluorescence resonance energy transfer analysis.
Anal Chem
; 85(3): 1468-74, 2013 Feb 05.
Article
em En
| MEDLINE
| ID: mdl-23276151
ABSTRACT
Analysis of protein dynamics using single-molecule fluorescence resonance energy transfer (smFRET) is widely used to understand the structure and function of proteins. Nonetheless, site-specific labeling of proteins with a pair of donor and acceptor dyes still remains a challenge. Here we present a general and facile method for site-specific dual labeling of proteins by incorporating two different, readily available, unnatural amino acids (p-acetylphenylalanine and alkynyllysine) for smFRET. We used newly evolved alkynyllysine-specific aminoacyl-tRNA synthetase/tRNA(UCA) and p-acetylphenylalanyl-tRNA synthetase/tRNA(CUA). The utility of our approach was demonstrated by analyzing the conformational change of dual-labeled calmodulin using smFRET measurements. The present labeling approach is devoid of major limitations in conventional cysteine-based labeling. Therefore, our method will significantly increase the repertoire of proteins available for FRET study and expand our ability to explore more complicated molecular dynamics.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA de Transferência
/
Proteínas
/
Transferência Ressonante de Energia de Fluorescência
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article