Visualization and Quantification of Sortase Activity at the Single

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Visualization and Quantification of Sortase Activity at the Single-molecule Level via Transpedidation-directed Intramolecular Förster Resonance Energy Transfer Yueying Li, Yong Yang, and Chun-yang Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03716 • Publication Date (Web): 11 Oct 2018 Downloaded from http://pubs.acs.org on October 13, 2018

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Analytical Chemistry

Visualization and Quantification of Sortase Activity at the Singlemolecule Level via Transpedidation-directed Intramolecular Förster Resonance Energy Transfer Yueying Li, † Yong Yang,*, § and Chun-yang Zhang*, † †

§

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. Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China

ABSTRACT: The Sortase-catalyzed coupling reaction is an efficient strategy to incorporate the chemically defined modification into the 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 transpedidation-directed intramolecular förster resonance energy transfer (FRET). This assay utilizes two cyanine dye-peptide conjugates, in which one carries a 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.aueus) 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 in-depth study of Sortases and for the discovery of anti-infective agents.

Bioorthogonal, chemoselective ligation and modification strategies have emerged as the essential tools in protein chemistry. The chemoenzymatic ligation / modiﬁcation can install the desired functional groups onto the proteins of interest in a controlled fashion. 1,2 The generated bio-conjugates have extensive applications in the biomedical field. 3-5 For example, the site-selective labeling of a protein with a fluorophore enables the monitoring of intracellular trafficking of a specific protein and its association with other biomolecules. The sitespecific attachment of anticancer drugs to antibodies enables the generation of uniform antibody–drug conjugates with high therapeutic index.6 One prominent chemo-enzymatic approach for protein engineering is based on the transpeptidation reaction that is catalyzed by SrtA from S.aueus.7 SrtA is a membrane-bound cysteine transpeptidase that catalyzes the covalent attachment of proteins containing sorting motif (LPXTG, where X is any amino acid) to the cell wall of Gram-positive bacteria. More specifically, the active-site cysteine of SrtA cleaves the Thr-Gly bond to form an acyl–enzyme intermediate. The intermediate is then resolved by an N-terminal pentaglycine amine nucleophile, resulting in the formation of an amide bond between threonine of LPXT* with the pentaglycine cross-bridge of branched lipid II.8 By means of this scheme, the SrtA catalyzes a number of virulence- and colonization-associated proteins to the cell wall. The SrtA-catalyzed “bio-click” strategy can be performed in vitro,9 and several

SrtA variants have been evolved to enhance the performance of the ligation reaction.10 The transpeptidase activity of SrtA has been harnessed for a variety of applications, including protein ligation in living cells,11 attaching fluorophores/drugs to the antibodies, cyclizing proteins, and in vivo protein labeling.12 To date, several methods have been developed to measure SrtA activity, such as fluorogenic peptide cleavage assay, yeast surface display (YSD)-based fluorescent assay, and protein-fragment complementation assay.13-15 However, each of these methods has its own limitations. For example, the fluorogenic peptide cleavage assay does not provide the information about the formation of transpeptidation product.16 In addition, the inner filter effect may lead to underestimation of the true kinetic parameters.13 The YSD-based method has limited sensitivity and is thus restricted to high-concentration SrtA.14 The protein-fragment complementation assays usually involve laborious procedures for the preparation of complementary protein segments.16 Therefore, new methods that can accurately measure SrtA activity with high sensitivity are in high demand. Single-molecule (SM) techniques have become important tools for investigating biological events with unprecedented resolution and sensitivity.17-19 In contrast to the ensemble measurements, single-molecule techniques have the capability to examine the heterogeneous populations in detail and to reveal the full probability distribution of certain biomolecules.

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Of the currently available single-molecule techniques, singlemolecule förster resonance energy transfer (smFRET) has been extensively used to study biological entities and interactions.20,21 The smFRET primarily utilizes the energy-transfer interaction between donor and acceptor fluorophores to probe the structural change of certain biomolecule and the interaction between two molecules. By monitoring the fluctuation of the donor and acceptor fluorescence signals in real time, smFRET may provide a variety of detailed information (e.g., population heterogeneity, transient intermediate state, parallel reaction pathway, and spontaneous stochastic behavior) that is difficult to obtain from ensemble measurement. The smFRET can be carried out through a confocal microscopy or a TIRF microscope. 21,22 Among them, the TIRF microscope exploits an evanescent field wave that is generated at the interface of two mediums to excite donor fluorophores for FRET.23 Generally, only the donor fluorophores adjacent to the surface (< 200 nm) can be excited while the rest remain dark. Accordingly, the TIRF microscope has the capacity to obtain highcontrast images of fluorophores with very low background. In addition, the TIRF-based imaging can monitor hundreds of single molecules simultaneously to acquire large amounts of data.20 The TIRF-based smFRET techniques have been widely used to answer questions about protein folding and binding, RNA dynamics and catalysis, gene transcription, membrane receptor interaction and oligomerization, and signal transduction.24-27 Herein, we take advantage of smFRET technique to develop a strategy that can accurately determine the kinetic parameter of SrtA. The assay relies on SrtA-catalyzed proximity of FRET pair and the subsequent smFRET detection via TIRF-based imaging. The proposed method can measure SrtA activity at the single-molecule level, and evaluate the inactivation of SrtA by a naturally occurring plant alkaloid. EXPERIMENTAL SECTION Materials. The SrtA protein was purchased from BPS Bioscience (CA, USA). The 1 M Tris-HCl (pH 7.5) was obtained from ThermoFisher Scientific (MA, USA). Berberine chloride was obtained from Sigma-Aldrich (MO, USA). Recombinant E.coli BirA protein (BirA) was purchased from Abcam (MA, USA). The Ubc9 (human) was purchased from Enzo Life Sciences (NY, USA). The T4 DNA ligase was obtained from Takara Biotechnology (Dalian, China). The S.aueus cell line was obtained from American Type Culture Collection (VA, USA). The CytoSelestTM Cell Adhesion Assasy Kit was purchased from Cell Biolabs (CA, USA). The brain heart infusion broth was purchased from BD-China (Shanghai, China). All peptides were synthesized by SciLight Biotechnology (Beijing, China) with purity greater than 95%. All solutions were prepared using the deionized water obtained from a MilliQ system (Millipore, USA). Reversed-Phase HPLC Assay. The reverse-phase HPLC (RP-HPLC) analysis was carried out at room temperature using an Agilent 1100 coupled with an UV detector. The peptide-dye conjugate GGGC-Cy5, Cy3-CLPETGG, Cy3CLPETGGGC-Cy5, and the GGGC-Cy5/Cy3-CLPETGG mixture were separated individually on a RP stainless-steel C18 analytical column (4.6 × 250 mm, 5 µm, Agela). The RPHPLC gradient was started at 10% solution B (0.05% TFA and 90% CH3CN in water), followed by increasing to 50% solution B at a flow rate of 1 mL/min for 50 min; and the solution A contained 0.05% TFA and 2% CH3CN in water. All mobile phases were filtered through a 0.22-µm filter (PALL-

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Gelman) and degassed using an ultrasonic bath prior to use. For SrtA-catalyzed ligation reaction, 106.8 μM GGGC-Cy5 and 75.7 μM Cy3-CLPETGG were mixed with 175 nM SrtA in 100 µL of SrtA reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, pH 7.5), and incubated at 37 ℃ for 2 h. The mixture was then heated at 95℃ for 20 min to terminate the reaction. The 20 µL of sample solution was injected into HPLC, and the peaks were detected by UV absorbance at 220 nm. The extent of reaction was quantified by comparing the peak area of the reaction product to that of a standard peptide solution (47.1 μM Cy3-CLPETGGGC-Cy5). SrtA-Mediated Transpeptidation Reaction and Fluorescence Measurement. The SrtA-catalyzed transpeptidation reaction was carried out at 37 ℃ in 20 µL of SrtA reaction buffer containing 4 µM GGGC-Cy5, 2 µM Cy3-CLPETGG, and 20 nM SrtA. The reaction mixture was diluted to 100 µL with the deionized water prior to the fluorescence measurement. The fluorescence signals were measured by an F-4600 spectrometer (Hitachi, Japan) with an excitation wavelength of 520 nm. The emission spectra were scanned from 540 to 750 nm, and the emission intensities at 560 nm (the maximum emission of Cy3) and 667 nm (the maximum emission of Cy5) were used for data analysis. The FRET efficiency E is calculated based on equation 1: = 1 −

(1)

where FDA is the fluorescence intensity of Cy3 in the presence of Cy5 acceptor, FD is the fluorescence intensity of Cy3 in the absence of Cy5 acceptor. Single-Molecule Detection and Data Analysis. To avoid the existence of multiple molecules in an individual spot, the reaction products were adequately diluted into the imaging buffer (1 mg/mL glucose oxidase, 0.4% (w/v) D-glucose, 0.04 mg/mL catalase, 50 µg/mL BSA, 67 mM glycine-KOH, 1 mg/mL trolox, 2.5 mM MgCl2, pH 9.4) prior to single molecule detection. For TIRF-based imaging, 10 µL of the diluted samples was pipetted onto the coverslip, and Cy3 was excited by a sapphire 561-nm laser (Coherent, USA). The emitted photons from Cy3 and Cy5 were collected by an oil immersion objective (NA 1.45, 100×, Olympus, Japan), and then separated and steered onto the two halves of an EMCCD camera (Ixon DU897, Andor Technology Plc., UK) by using beam splitter (Optosplit II, Andor Technology Plc., UK). Captured images were analyzed using the ImageJ software. Generally, a region of 400 × 400 pixels was selected for Cy5 counting, and only spots with signal intensities that are at least 1.5-fold above the background were used for data analysis. Meanwhile, the numbers of Cy3 were counted and used as an internal control to correct the numbers of Cy5. The singlemolecule photobleaching experiment was carried out as previously described.28,29 In brief, the diluted reaction mixture was continuously excited by either a 561-nm laser (34 mW) or a 640-nm laser (20 mW), and the live images were recorded at a high-frequency with an exposure time of 60 ms. The 300 frames were used for data analysis. All the measurements were performed at room temperature. Inhibition of SrtA Activity by Berberine Chloride. Berberine chloride was dissolved in DMSO to make a stock solution (1 mg/mL). Different-concentration berberine chloride was mixed with 20 nM SrtA in SrtA reaction buffer and incubated at 37 ℃ for 10 min. Then 4 µM GGGC-Cy5 and 2 µM Cy3-CLPETGG were added to the mixture and incubated at


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Analytical Chemistry 37 ℃ for 2 h. The relative activity (RA) of SrtA was determined using equation 2: RA =

× 100%

(2)

where N0 is the number of Cy5 counts in the absence of SrtA, Nt is the number of Cy5 counts in the presence of 20 nM SrtA, and Ni is the number of Cy5 counts in the presence of both 20 nM SrtA and berberine chloride. The IC50 value was calculated by plotting the RA values versus the concentration of berberine chloride. Kinetic Assay of SrtA. The Km value of SrtA for Cy3CLPETGG was determined by using different-concentration Cy3-CLPETGG (2, 10, 50, 250, 500, 1000, 2000 µM) while keeping the concentration of GGGC-Cy5 constant (4 mM). The Km value for GGGC-Cy5 was determined by using different-concentration GGGC-Cy5 (1.6, 8, 40, 200, 1000, 2000, 4000, 8000 µM) while keeping the concentration of Cy3CLPETGG constant (1 mM). The Cy3-CLPETGG/GGGCCy5 mixture was incubated with 20 nM SrtA at 37 °C for 10 min, and the transpeptidation reaction was terminated by heat inactivation of SrtA. The reaction products were subjected to single-molecule detection as described above, and the Km values were calculated by fitting the obtained initial velocities to equation 3. V =

×

(3)

where V0 is the initial velocity, Vmax is the maximum reaction velocity, [S] is the concentration of dye-peptide conjugate, and Km is the Michaelis constant. Measurement of SrtA Activity in S. aureus Cells. The S. aureus cells were cultured in brain heart infusion (BHI) broth and harvested until the bacterial suspension reached an optical density of 0.6 at 600 nm (OD600). The collected S. aureus cells were resuspended in SrtA reaction buffer. Subsequently, 4 µM GGGC-Cy5 and 2 µM Cy3-CLPETGG were mixed with 2 µL of S. aureus cells suspension, and incubated at 37 ℃ for 2 h to carry out the transpedidation reaction. The S. aureus cells were removed via centrifugation, and the supernatant was subjected to single molecule detection following approximate dilution. For the inhibition of intracellular SrtA, variableconcentration berberine chloride (1.25, 2.5, 5, 10, 20, 40, 80 µg/mL) was added to S. aureus cells suspension, followed by incubation with GGGC-Cy5 and Cy3-CLPETGG. The reaction mixtures were subsequently subjected to single molecule detection as previously described. Fibrinogen-Binding Assay. The S. aureus cells from 800 µL of BHI broth were collected via centrifugation and resuspended in 200 µL of phosphate-buffered saline (PBS). The obtained S. aureus cell suspensions were added to the wells of fibrinogen-coated 48-well microtiter plate (Cell Biolabs, CA, USA) and incubated at 37 °C for 90 min. After removing the solution, the wells were washed four times with 250 µL of PBS. The S. aureus cells that bound to fibrinogen were stained with 200 µL of cell stain solution for 10 min at room temperature. After discarding the cell stain solution, the wells were washed with 250 µL of PBS for five times. Subsequently, 200 µL of extraction solution was added to each well and incubated on an orbital shaker for 10 min. The absorbance at 560 nm was measured using a Flex Station 3 (Molecular Devices) after transferring 150 µL of extracted solution to a 96-well microtiter plate. Triplicate measurements were taken for each data point.

Growth Curve Analysis of S. aureus cells in the Presence of Berberine Chloride. The growth curve analysis was performed in 96-well black plate (Fisher Scientific) in a final volume of 100 µL. Briefly, the overnight-cultured S. aureus cells were 1:15 diluted into fresh BHI medium containing variable-concentration berberine chloride (0, 20, 40, or 80 µg/mL). The S. aureus cells were continuously cultured with shaking for 12 h. The absorbance at 600 nm was measured per 30 min using a Flex Station 3 (Molecular Devices). The data are presented as the means of triplicate measurements. RESULTS AND DISCUSSION

Scheme 1. (A) Principle of the SrtA-catalyzed transpedidation reaction for the formation of chimera Cy3CLPETGGGC-Cy5. (B) Schematic illustration of transpedidation-directed smFRET for SrtA assay. This assay involves three steps: (i) transpedidation reaction, (ii) intramolecular FRET, and (iii) single-molecule detection. The proposed assay is a signal-on fluorescence measurement that involves three sequential steps: (i) the generation of FRET-based chimeras via Sortase-catalyzed transpedidation reaction, (ii) the illumination of acceptor fluorophores by means of intramolecular FRET, and (iii) the single-molecule detection of FRET signal via TIRF-based imaging (Scheme 1). In the presence of SrtA, the SrtA initiates the transpedidation reaction to bring the donor and the acceptor into close proximity, which subsequently leads to illumination of acceptor fluorophores by means of smFRET. Conversely, in the absence of SrtA, the donor and acceptor fluorophores remain physically separated, resulting in no FRET signal. Accordingly, the SrtA activity can be quantitatively measured at the single-molecule level by counting the number of the illuminated acceptor fluorophores. As a proof of concept, we demonstrated the quantitative measurement of SrtA activity both in vitro and in S.aueus cells.

Figure 1. Characterization of SrtA-catalyzed transpedidation reaction. (A) HPLC analysis of SrtA-catalyzed ligation reaction. Five different samples including (a) Cy3-CLPETGG + GGGCCy5 + SrtA, (b) Cy3-CLPETGG + GGGC-Cy5, (c) Cy3-



CLPETGGGC-Cy5, (d) GGGC-Cy5, (e) Cy3-CLPETGG) were subjected to HPLC analysis. The peaks correspond to Cy3CLPETGG (green), GGGC-Cy5 (red), and Cy3-CLPETGGGCCy5 (yellow) were indicated by inverted triangles. (B) Emission spectra of Cy3-CLPETGG/GGGC-Cy5 mixture with (blue) and without (red) SrtA treatment (λex = 520 nm). The fluorescence emission spectra of GGGC-Cy5 under the excitation of 520 nm (black) and 560 nm (dark cyan) were shown as well.

Two fluorescently labeled peptides, Cy3-CLPETGG and GGGC-Cy5, served as the substrate of SrtA. The SrtA may catalyze the fusion of them to form a chimera Cy3CLPETGGGC-Cy5. To verify whether SrtA can efficiently ligate two segments or not, the Cy3-CLPETGG / GGGC-Cy5 mixture was incubated with SrtA, followed by reverse-phase high performance liquid chromatography (HPLC) analysis. As shown in Figure 1A, the SrtA-catalyzed ligation reaction leads to the generation of a product (t = 47 min) that is well matched to that of the synthesized Cy3-CLPETGGGC-Cy5. This result indicates that the SrtA can recognize Cy3-CLPETGG/GGGCCy5 pair and catalyze them to form a dual-labeled chimera. We further measured the emission spectra of Cy3-CLPETGG / GGGC-Cy5 mixture after treating with SrtA. The incubation of SrtA with Cy3-CLPETGG/GGGC-Cy5 mixture leads to simultaneous emission spectrum of Cy3 and Cy5, in contrast to only the Cy3 signal in the absence of SrtA (Figure 1B). It should be noted that Cy5 was illuminated by intracellular FRET from Cy3 to Cy5 because the excitation wavelength of 520 nm produced a negligible fluorescence for GGGC-Cy5. To achieve a better assay performance, the reaction time and the binding stoichiometry between two dye-peptide conjugates were optimized. Upon addition of SrtA, the amount of ligation product (i.e., Cy3-CLPETGGGC-Cy5) increased gradually over time and reached its maximum at 120 min (see supporting information, Figure S1), thus 120 min is selected as the time for transpeptidation reaction. The fluorescence intensity of Cy5 and the FRET efficiency are strongly associated with the ratio of GGGC-Cy5 to Cy3-CLPETGG. The maximum values were obtained at the ratio of 2:1 (see supporting information, Figure S2). We further investigated whether the FRET signal within the formed chimera can be detected by TIRF microscope. As shown in Figure 2, the incubation of SrtA with Cy3-CLPETGG/GGGC-Cy5 mixture results in simultaneous fluorescence signals of Cy3 and Cy5 (Figures 2d and 2e). In contrast, only Cy3 fluorescence signal is observed in the absence of SrtA (Figure 2a). The Cy5 fluorescence signals perfectly co-localized with the Cy3 fluorescence signals (Figure 2f), further demonstrating that the emission of Cy5 is induced by intramolecular FRET from Cy3 to Cy5. In addition, both Cy3 and Cy5 fluorescence spots exhibit single-step photobleaching (see supporting information, Figure S3), suggesting that the observed spots contain single molecule. Therefore, the TIRF-based single-molecule detection is amenable to analyze the transpeptidase activity of Sortase.

Figure 2. Fluorescence images of Cy3-CLPETGG/GGGC-Cy5 mixture treated without (a-c) and with (d-f) SrtA. The green color (a and d) represents the signal of Cy3, the red color (e) represents the signal of Cy5. The fluorescent spots are shown as pseudocolors. Scale bar is 5 μm.

Under the optimal conditions, we evaluated the specificity of the assay. The Cy3-CLPETGG/GGGC-Cy5 mixture was incubated with SrtA, together with Ubc9, BirA, and T4 DNA ligase as well. The mixture without enzyme treatment was used as the negative control. The resulting mixtures were imaged by TIRF microscope to record the number of Cy5 counts. The addition of SrtA results in large amounts of Cy5 counts, whereas the other ligases display negligible Cy5 counts, similar to that of negative control (Figure 3A and Figure S4). This result indicates that the proposed assay is specific to SrtA. We further investigated the sensitivity of the assay by measuring the increment of Cy5 counts in response to variableconcentration SrtA. As shown in Figure 3B, the number of Cy5 counts enhances substantially with increasing concentration of SrtA from 12.5 pM to 20 nM. A linear correlation is obtained between Cy5 counts and the SrtA concentration in the range from 12.5 to 400 pM (inset of Figure 3B). The obtained correlation equation is N = 81.14 + 3.59 C (R2 = 0.995), where N refers to the number of Cy5 counts and C is the concentration of SrtA (pM). The limit of detection (LOD) is calculated to be 7.08 pM by evaluating the average response of the negative control plus 3 times standard deviation. Notably, the LOD of the proposed assay is much lower than those of reported methods (840 nM 13 and 0.16 nM 15). The improved sensitivity may be attributed to the high sensitivity of singlemolecule detection.30 Moreover, the intramolecular FRET allows the assay to be carried out in a homogeneous format without the involvement of any washing and separation procedures, making the assay quite simple.

Figure 3. (A) Measurement of Cy5 counts after incubating Cy3CLPETGG/GGGC-Cy5 mixture with 20 nM SrtA, 20 nM Ubc9,


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Analytical Chemistry 20 nM BirA, and 20 nM T4 DNA ligase, respectively. The Cy3CLPETGG/GGGC-Cy5 mixture without any treatment was used as the control. (B) Variance of Cy5 counts in response to different-concentration SrtA. Inset: the Cy5 counts show a linear correlation with the SrtA concentration in the range from 12.5 to 400 pM. Error bars show the standard deviation of three independent experiments.

indicate that the proposed assay is versatile and amenable to characterize SrtA-targeted agents.

We further investigated whether this strategy can be used to accurately measure the kinetic parameters of SrtA. To do this, we defined the rate of transpedidation reaction as the increment of Cy5 counts per minute. The initial reaction rates were fitted to Michaelis-Menten equation to determine the Michaelis constant Km. The Km values of SrtA for Cy3CLPETGG and GGGC-Cy5 were 218.9 µM and 2212.4 µM, respectively (Figure 4). It is noteworthy that the obtained Km values are consistent with those obtained by the standard HPLC assay,10 suggesting that the single-molecule detection performed at extremely low concentration of substrates can efficiently eliminate the inner filter effect and the collisional quenching which occur in conventional ensemble fluorescence measurement.31 Figure 5. Inhibition of SrtA activity in vitro and in S.aueus cells by berberine chloride. (A) Chemical structure of berberine chloride. (B) Relative activity of purified SrtA in response to different-concentration berberine chloride. (C) Berberine chloride inhibits the adhesion of S. aureus cells to fibrinogen. (D) Variance of Cy5 counts as a function of the concentration of berberine chloride administrated to S. aureus cells.

Figure 4. Measurement of kinetic parameters of SrtA-catalyzed transpedidation reaction. (A) Measurement of SrtA’s Michaelis constant for Cy3-CLPETGG. (B) Measurement of SrtA’s Michaelis constant for GGGC-Cy5. Error bars show the standard deviation of three independent experiments.

The S. aureus strain has acquired resistance against a variety of antibiotics, such as fusidic acid, rifampicin, mupiricin, methicillin, and vancomycin.32 Blocking SrtA activity represents a promising way to prevent infection caused by antibiotic-resistant S. aureus.33,34 To demonstrate the feasibility of the proposed method for the characterization of SrtA-targeted inhibitors, we employed berberine chloride (Figure 5A), a natural plant alkaloid found in a wide variety of traditional Chinese herbs, as a model inhibitor to block SrtA activity. As shown in Figure 5B, the berberine chloride displayed a dosedependent inhibition of SrtA activity. The estimated half maximal inhibitory concentration (IC50) for berberine chloride is 8.4 µg/mL, consistent with the reported value of 8.7 µg/mL. 35 The berberine chloride was further used to inhibit SrtA activity in S. aureus cells. As shown in Figure 5C, the administration of berberine chloride to S. aureus cells results in the decrease of adherence capability toward fibrinogen-coated surface as compared to that of untreated cells. Similarly, the increase of berberine chloride concentration from 1.25 to 80 µg/mL leads to substantially decrease of Cy5 counts (Figure 5D and Figure S5). Nevertheless, the berberine chloride does not inhibit the growth of S. aureus cells even at a concentration of up to 80 µg/mL (see supporting information, Figure S6), suggesting that the SrtA-targeted inhibitor may function as a potential anti-virulence drug. Taken together, these results

CONCLUSIONS In summary, we have developed a straightforward and homogenous method to determine SrtA activity at the singlemolecule level. The assay involves the transpedidationmediated proximity of FRET pair and the subsequent singlemolecule counting. This assay can effectively overcome the inner filter effect that occurs in ensemble fluorescence measurement, and accurately determine the kinetic parameter of SrtA. In addition, this assay can measure SrtA with a detection limit of 7.08 pM, much lower than those of the reported assays. This assay can also be used to characterize SrtA inhibitors both in vitro and in S.aueus cells. Importantly, this assay is highly versatile, and it can be extended to measure other Sortases and peptide-peptide bond-forming enzymes (e.g., subtiligase, peptiligase, transglutaminase, butelase) by adopting appropriate recognition sequence, thereby providing a powerful tool for in-depth study of Sortase and for the discovery of anti-virulence drugs.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. The time course of SrtA-catalyzed transpeptidation reaction, the optimization of the ratio between two dye-peptide conjugates, the photobleaching traces of individual Cy3 and Cy5 fluorescence spots, and the growth curve of S. aueus cells treated with different-concentration berberine chloride (Figures S1−S6) (PDF).

AUTHOR INFORMATION Corresponding Author



* E-mail: [email protected]; Tel.: +86 0531-86186033; Fax: +86 0531-82615258. E-mail: [email protected]; Fax: +86 0755-86392229; Tel: +86 0755-86585240.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grant Nos. 21675168, 21325523, 21527811, and 21735003), the Natural Science Foundation of Shenzhen City (Grant Nos. JCYJ20170818161859427, GJHS20170314161339765), the “Guangdong TeZhi Plan” Youth Talent of Science and Technology, and the Youth Innovation Promotion Association of the Chinese Academy of Sciences (No. 2016323). REFERENCES (1) Rosen, C. B.; Francis, M. B. Targeting the N terminus for siteselective protein modification. Nat. Chem. Biol. 2017, 13, 697-705. (2) Rabuka, D. Chemoenzymatic methods for site-specific protein modification. Curr. Opin. Chem. Biol. 2010, 14, 790-796. (3) Mullard, A. Maturing antibody-drug conjugate pipeline hits 30. Nat. Rev. Drug Discov. 2013, 12, 329-332. (4) Glasgow, J. E.; Salit, M. L.; Cochran, J. R. In Vivo Site-Specific Protein Tagging with Diverse Amines Using an Engineered Sortase Variant. J. Am. Chem. Soc. 2016, 138, 7496-7499. (5) Tang, F.; Wang, L. X.; Huang, W. Chemoenzymatic synthesis of glycoengineered IgG antibodies and glycosite-specific antibody-drug conjugates. Nat. Protoc. 2017, 12, 1702-1721. (6) Beck, A.; Goetsch, L.; Dumontet, C.; Corvaia, N. Strategies and challenges for the next generation of antibody-drug conjugates. Nat. Rev. Drug Discov. 2017, 16, 315-337. (7) Antos, J. M.; Truttmann, M. C.; Ploegh, H. L. Recent advances in sortase-catalyzed ligation methodology. Curr. Opin. Struct. Biol. 2016, 38, 111-118. (8) Paterson, G. K.; Mitchell, T. J. The biology of Gram-positive sortase enzymes. Trends Microbiol. 2004, 12, 89-95. (9) Popp, M. W.; Antos, J. M.; Grotenbreg, G. M.; Spooner, E.; Ploegh, H. L. Sortagging: a versatile method for protein labeling. Nat. Chem. Biol. 2007, 3, 707-708. (10) Chen, I.; Dorr, B. M.; Liu, D. R. A general strategy for the evolution of bond-forming enzymes using yeast display. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 11399-11404. (11) Strijbis, K.; Spooner, E.; Ploegh, H. L. Protein ligation in living cells using sortase. Traffic 2012, 13, 780-789. (12) Wu, Q.; Ploegh, H. L.; Truttmann, M. C. Hepta-Mutant Staphylococcus aureus Sortase A (SrtA7m) as a Tool for in Vivo Protein Labeling in Caenorhabditis elegans. ACS Chem. Biol. 2017, 12, 664-673. (13) Kruger, R. G.; Dostal, P.; McCafferty, D. G. Development of a high-performance liquid chromatography assay and revision of kinetic parameters for the Staphylococcus aureus sortase transpeptidase SrtA. Anal. Biochem. 2004, 326, 42-48. (14) Wu, L.; Li, H.; Tang, T. A Novel Yeast Surface Display Method for Large-Scale Screen Inhibitors of Sortase A. Bioengineering 2017, 4, 6. (15) Zhang, J.; Wang, M.; Tang, R.; Liu, Y.; Lei, C.; Huang, Y.; Nie, Z.; Yao, S. Transpeptidation-Mediated Assembly of Tripartite Split Green Fluorescent Protein for Label-Free Assay of Sortase Activity. Anal. Chem. 2018, 90, 3245-3252.

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Analytical Chemistry

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7

Visualization and Quantification of Sortase Activity at the Single

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