TY - JOUR
T1 - Development of Diubiquitin-Based FRET Probes to Quantify Ubiquitin Linkage Specificity of Deubiquitinating Enzymes
AU - Geurink, Paul P.
AU - Van Tol, Bianca D.M.
AU - Van Dalen, Duco
AU - Brundel, Paul J.G.
AU - Mevissen, Tycho E.T.
AU - Pruneda, Jonathan N.
AU - Elliott, Paul R.
AU - Van Tilburg, Gabriëlle B.A.
AU - Komander, David
AU - Ovaa, Huib
N1 - Publisher Copyright:
© 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
PY - 2016/5/3
Y1 - 2016/5/3
N2 - 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.
AB - 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.
KW - FRET
KW - deubiquitinating enzymes
KW - native chemical ligation
KW - solid-phase synthesis
KW - ubiquitin conjugates
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U2 - 10.1002/cbic.201600017
DO - 10.1002/cbic.201600017
M3 - Article
C2 - 26996281
AN - SCOPUS:84962623766
SN - 1439-4227
VL - 17
SP - 816
EP - 820
JO - ChemBioChem
JF - ChemBioChem
IS - 9
ER -