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Real-Time and Label-Free Measurement of Deubiquitinase Activity with a MspA Nanopore.

ABSTRACT: Covalently attaching ubiquitin (Ub) to cellular proteins as a post-translational modification can result in altered function of modified proteins. Enzymes regulating Ub as a post-translational modification, such as ligases and deubiquitinases, are challenging to characterize in part due to the low throughput of in-vitro assays. Single-molecule nanopore based assays have the advantage of detecting proteins with high specificity and resolution, and in a label-free, real-time fashion. Here we demonstrate the use of a MspA nanopore for discriminating and quantifying Ub proteins. We further applied the MspA pore to measure the Ub-chain disassembly activity of UCH37, a proteasome associated deubiquitinase. The implementation of this MspA system into nanopore arrays could enable high throughput characterizations of unknown deubiquitinases as well as drug screening against disease related enzymes.

SUBMITTER: Shorkey SA

PROVIDER: S-EPMC8416795 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Real-Time and Label-Free Measurement of Deubiquitinase Activity with a MspA Nanopore.

Shorkey Spencer A SA Du Jiale J Pham Ryan R Strieter Eric R ER Chen Min M

Chembiochem : a European journal of chemical biology 20210616 17

Covalently attaching ubiquitin (Ub) to cellular proteins as a post-translational modification can result in altered function of modified proteins. Enzymes regulating Ub as a post-translational modification, such as ligases and deubiquitinases, are challenging to characterize in part due to the low throughput of in-vitro assays. Single-molecule nanopore based assays have the advantage of detecting proteins with high specificity and resolution, and in a label-free, real-time fashion. Here we demon ...[more]

PMID: 34060221

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