Development of Diubiquitin-Based FRET Probes To Quantify Ubiquitin Linkage Specificity of Deubiquitinating Enzymes

Overview

Journal Chembiochem

Specialty Biochemistry

Date 2016 Mar 22

PMID 26996281

Citations 28

Authors

Paul P Geurink

Bianca D M van Tol

Duco van Dalen

Paul J G Brundel

Tycho E T Mevissen

Jonathan N Pruneda

Paul R Elliott

Gabrielle B A van Tilburg

David Komander

Huib Ovaa

Affiliations

Soon will be listed here.

Abstract

Deubiquitinating enzymes (DUBs) are proteases that fulfill crucial roles in the ubiquitin (Ub) system, by deconjugation of Ub from its targets and disassembly of polyUb chains. The specificity of a DUB towards one of the polyUb chain linkages largely determines the ultimate signaling function. We present a novel set of diubiquitin FRET probes, comprising all seven isopeptide linkages, for the absolute quantification of chain cleavage specificity of DUBs by means of Michaelis-Menten kinetics. Each probe is equipped with a FRET pair consisting of Rhodamine110 and tetramethylrhodamine to allow the fully synthetic preparation of the probes by SPPS and NCL. Our synthetic strategy includes the introduction of N,N'-Boc-protected 5-carboxyrhodamine as a convenient building block in peptide chemistry. We demonstrate the value of our probes by quantifying the linkage specificities of a panel of nine DUBs in a high-throughput manner.

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