Visualization and Quantification of Sortase Activity at the Single-Molecule Level via Transpeptidation-Directed Intramolecular Förster Resonance Energy Transfer

Abstract

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 selfquenching. Herein, we develop a new method to visualize and quantify sortase A (SrtA) activity at the single-molecule level by using transpeptidation-directed intramolecular Forster 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.

Chemical Biology of Sortase A Inhibition: A Gateway to Anti-infective Therapeutic Agents

“…The assay designed by Zhang and co-workers exploited the use of FRET between cyanine dye−peptide conjugates, Cy3-CLPETGG and GGGG-Cy5. 106 In contrast to Dabcyl-EDANS and Abz-DNP-based peptide cleavage assays, this assay relies on SrtA-catalyzed chemical ligation of peptides bringing donor (Cy3) and acceptor (Cy5) in proximity to each other. This results in enhanced fluorescence emission of Cy5 which can be quantified by fluorescence imaging.…”

“…The nature of data on the biological activity of a compound is dependent on the type of the biological assay performed. Many conventional assays such as fluorescence resonance energy transfer (FRET), ,,,,− high-performance liquid chromatography (HPLC), ,, cell adhesion, , clumping, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), , and fluorescein incorporation assay, have been employed and reported in the literature to assess the SrtA inhibitory activity of inhibitors. It is useful to briefly survey these different techniques since it benefits the medicinal chemistry community.…”

“…Recently, more sophisticated fluorescence-based assays have been developed to measure the SrtA inhibition potency of a test compound. The assay designed by Zhang and co-workers exploited the use of FRET between cyanine dye–peptide conjugates, Cy3-CLPETGG and GGGG-Cy5 . In contrast to Dabcyl-EDANS and Abz-DNP-based peptide cleavage assays, this assay relies on SrtA-catalyzed chemical ligation of peptides bringing donor (Cy3) and acceptor (Cy5) in proximity to each other.…”

Label- and enzyme-free plasmon-enhanced single molecule fluorescence detection of HIV DNA fragments based on a catalytic hairpin assembly

“…Compared to the traditional fluorescence detection methods, which measure the overall state of fluorescent molecules, single molecule detection technology can reveal heterogeneous fluorescent molecules and the distribution of biomolecules in detail. 38,39 However, due to the low fluorescence intensity and easy photobleaching of single fluorescent molecules, the accuracy of single molecule fluorescence analysis will be affected and its application will be limited. Therefore, it is necessary to improve the luminance and optical stability of the fluorophores.…”