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Visualization and Quantification of Sortase Activity at the Single-Molecule Level via Transpeptidation-Directed Intramolecular Förster Resonance Energy Transfer.
Li, Yueying; Yang, Yong; Zhang, Chun-Yang.
Afiliación
  • Li Y; College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals , Shandong Normal University , Jinan 250014 , China.
  • Yang Y; Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology , Chinese Academy of Sciences , Shenzhen 518055 , China.
  • Zhang CY; College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals , Shandong Normal University , Jinan 250014 , China.
Anal Chem ; 90(21): 13007-13012, 2018 11 06.
Article en En | MEDLINE | ID: mdl-30350606
The sortase-catalyzed coupling reaction is an efficient strategy to incorporate chemically defined modifications into proteins of interest. Despite its widespread applications in protein chemistry, the conventional bulk fluorescence measurement is not sufficient to characterize sortase due to the fluorescence inner filter effect-induced self-quenching. Herein, we develop a new method to visualize and quantify sortase A (SrtA) activity at the single-molecule level by using transpeptidation-directed intramolecular Förster resonance energy transfer (FRET). This assay utilizes two cyanine dye-peptide conjugates, in which one carries an LPXTG motif and a donor fluorophore while the other harbors an oligoglycine nucleophile and an acceptor fluorophore as the substrate of SrtA. The presence of SrtA catalyzes the fusion of two conjugates and allows for the occurrence of intramolecular FRET. The FRET signal is recorded at the single-molecule level via total internal reflection fluorescence (TIRF)-based imaging. The proposed assay not only can accurately determine the kinetic parameters of SrtA but also can characterize the inhibition of SrtA activity by berberine chloride both in vitro and in Staphylococcus aureus ( S. aureus) cells. Moreover, the assay is very specific, and it can sensitively measure SrtA down to 7.08 pM, which is much lower than most of the reported methods. This strategy may provide a valuable tool for an in-depth study of sortases and for the discovery of anti-infective agents.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas Bacterianas / Cisteína Endopeptidasas / Aminoaciltransferasas / Transferencia Resonante de Energía de Fluorescencia / Imagen Individual de Molécula Idioma: En Revista: Anal Chem Año: 2018 Tipo del documento: Article País de afiliación: China Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas Bacterianas / Cisteína Endopeptidasas / Aminoaciltransferasas / Transferencia Resonante de Energía de Fluorescencia / Imagen Individual de Molécula Idioma: En Revista: Anal Chem Año: 2018 Tipo del documento: Article País de afiliación: China Pais de publicación: Estados Unidos