Simultaneous Measurement of SUMOylation using SNAP/CLIP‐Tag‐Mediated Translation at the Single‐Molecule Level

Yang, Yong; Zhang, Chunyang

doi:10.1002/anie.201206695

Cited by 20 publications

(7 citation statements)

References 43 publications

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“…Notably, the LOD of the proposed assay is much lower than those of reported methods (840 nM and 0.16 nM). The improved sensitivity may be attributed to the high sensitivity of single-molecule detection . Moreover, 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.…”

Section: Resultsmentioning

confidence: 99%

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

Yang

Zhang

2018

Anal. Chem.

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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.

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Section: Resultsmentioning

confidence: 99%

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

Yang

Zhang

2018

Anal. Chem.

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“…Such specificity may be obtained using display techniques to select Ub and Ubl variants, as pioneered by Ernst et al [92] or synthetic designer reagents. Intriguing recent approaches furthermore include the SNAP/CLIP tag and the tetracysteine tag [93][94][95]. As the field and drug development efforts come of age, the toolbox of techniques reagents and inhibitors will steadily grow.…”

Section: What Other Techniques and Assays Are Currently Missing?mentioning

confidence: 99%

Probing ubiquitin and SUMO conjugation and deconjugation

Ovaa

Vertegaal

2018

Biochemical Society Transactions

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Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins including small Ubl modifier (SUMO) are small proteins which are covalently linked to target proteins to regulate their functions. In this review, we discuss the current state of the art and point out what we feel this field urgently needs in order to delineate the wiring of the system. We discuss what is needed to unravel the connections between different components of the conjugation machineries for ubiquitylation and SUMOylation, and to unravel the connections between the conjugation machineries and their substrates. Chemical probes are key tools to probe signal transduction by these small proteins that may help understand their action. This rapidly moving field has resulted in various small molecules that will help us to further understand Ub and SUMO function and that may lead to the development of new drugs.

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“…荧光标记的甲基化结合蛋白(methyl-CpGbinding domain, MBD) [81] 和甲基转移酶(DNA methyltransferase, MTase) [82] 常被用于研究甲基化修饰. Kim 等 [48] [51] (网络版彩图)…”

Section: 生物分子修饰unclassified

Advance in quantitative single-molecule detection

Juan¹,

Wang²,

Zhang³

2017

Sci. Sin.-Chim

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Simultaneous Measurement of SUMOylation using SNAP/CLIP‐Tag‐Mediated Translation at the Single‐Molecule Level

Cited by 20 publications

References 43 publications

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

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

Probing ubiquitin and SUMO conjugation and deconjugation

Advance in quantitative single-molecule detection

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