title: Development of Diubiquitin-Based FRET Probes To Quantify Ubiquitin Linkage Specificity of Deubiquitinating Enzymes.
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.
authors.affiliation: Department of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066, CX, Amsterdam, The Netherlands. p.geurink@nki.nl.
authors.forename: Paul P
authors.initials: PP
authors.lastname: Geurink
authors.affiliation: Department of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066, CX, Amsterdam, The Netherlands.
authors.forename: Bianca D M
authors.initials: BD
authors.lastname: van Tol
authors.affiliation: Department of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066, CX, Amsterdam, The Netherlands.
authors.forename: Duco
authors.initials: D
authors.lastname: van Dalen
authors.affiliation: Department of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066, CX, Amsterdam, The Netherlands.
authors.forename: Paul J G
authors.initials: PJ
authors.lastname: Brundel
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, FrancisCrick Avenue, Cambridge, CB2 0QH, UK.
authors.forename: Tycho E T
authors.initials: TE
authors.lastname: Mevissen
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, FrancisCrick Avenue, Cambridge, CB2 0QH, UK.
authors.forename: Jonathan N
authors.initials: JN
authors.lastname: Pruneda
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, FrancisCrick Avenue, Cambridge, CB2 0QH, UK.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Elliott
authors.affiliation: Department of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066, CX, Amsterdam, The Netherlands.
authors.forename: Gabriëlle B A
authors.initials: GB
authors.lastname: van Tilburg
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, FrancisCrick Avenue, Cambridge, CB2 0QH, UK.
authors.forename: David
authors.initials: D
authors.lastname: Komander
authors.affiliation: Department of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066, CX, Amsterdam, The Netherlands. h.ovaa@nki.nl.
authors.forename: Huib
authors.initials: H
authors.lastname: Ovaa
mesh_terms: D002851:Chromatography, High Pressure Liquid; D000072017:Deubiquitinating Enzymes / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D005456:Fluorescent Dyes; D025801:Ubiquitin; D000072017:Deubiquitinating Enzymes
keywords: FRET; deubiquitinating enzymes; native chemical ligation; solid-phase synthesis; ubiquitin conjugates