Dataset Information

Synthesis of Poly-Ubiquitin Chains Using a Bifunctional Ubiquitin Monomer.

ABSTRACT: An optimized large scale and highly reproducible route to orthogonally protected ?-thiolysine is reported. Its utility in the synthesis of bifunctional ubiquitin monomers is demonstrated. These ubiquitin synthons are employed in polymerization reactions giving access to synthetic poly-ubiquitin chains of defined linkage.

SUBMITTER: van der Heden van Noort GJ

PROVIDER: S-EPMC5735377 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Publications

Synthesis of Poly-Ubiquitin Chains Using a Bifunctional Ubiquitin Monomer.

van der Heden van Noort Gerbrand J GJ Kooij Raymond R Elliott Paul R PR Komander David D Ovaa Huib H

Organic letters 20171127 24

An optimized large scale and highly reproducible route to orthogonally protected γ-thiolysine is reported. Its utility in the synthesis of bifunctional ubiquitin monomers is demonstrated. These ubiquitin synthons are employed in polymerization reactions giving access to synthetic poly-ubiquitin chains of defined linkage. ...[more]

PMID: 29172548

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Project description:Nanopore sensing involves an electrophoretic transport of analytes through a nanoscale pore, permitting label-free sensing at the single-molecule level. However, to date, the detection of individual small proteins has been challenging, primarily due to the poor signal/noise ratio that these molecules produce during passage through the pore. Here, we show that fine adjustment of the buffer pH, close to the isoelectric point, can be used to slow down the translocation speed of the analytes, hence permitting sensing and characterization of small globular proteins. Ubiquitin (Ub) is a small protein of 8.5 kDa, which is well conserved in all eukaryotes. Ub conjugates to proteins as a posttranslational modification called ubiquitination. The immense diversity of Ub substrates, as well as the complexity of Ub modification types and the numerous physiological consequences of these modifications, make Ub and Ub chains an interesting and challenging subject of study. The ability to detect Ub and to identify Ub linkage type at the single-molecule level may provide a novel tool for investigation in the Ub field. This is especially adequate because, for most ubiquitinated substrates, Ub modifies only a few molecules in the cell at a given time. Applying our method to the detection of mono- and poly-Ub molecules, we show that we can analyze their characteristics using nanopores. Of particular importance is that two Ub dimers that are equal in molecular weight but differ in 3D structure due to their different linkage types can be readily discriminated. Thus, to our knowledge, our method offers a novel approach for analyzing proteins in unprecedented detail using solid-state nanopores. Specifically, it provides the basis for development of single-molecule sensing of differently ubiquitinated substrates with different biological significance. Finally, our study serves as a proof of concept for approaching nanopore detection of sub-10-kDa proteins and demonstrates the ability of this method to differentiate among native and untethered proteins of the same mass.

Dataset Information

Synthesis of Poly-Ubiquitin Chains Using a Bifunctional Ubiquitin Monomer.

Publications

Synthesis of Poly-Ubiquitin Chains Using a Bifunctional Ubiquitin Monomer.

Similar Datasets

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