Search results for: 5_CARBOXYRHODAMINE 110_PEO8_PROPIONATE
#26996281 
2016/03/21
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Development of Diubiquitin-Based FRET Probes To Quantify Ubiquitin Linkage Specificity of Deubiquitinating Enzymes.
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.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, Gabriëlle B A van Tilburg, David Komander, Huib Ovaa
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5 G1 module200 ugOne 96-Well Microplate KiOne 96-Well Strip Micropl 6 ml Ready-to-use 25 G430 Tests / KitML
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Structural exploration and Förster theory modeling for the interpretation of gas-phase FRET measurements: Chromophore-grafted amyloid-β peptides.
The distance-dependence of excitation energy transfer, e.g., being described by Förster theory (Förster resonance energy transfer (FRET)), allows the use of optical techniques for the direct observation of structural properties. Recently, this technique has been successfully applied in the gas phase. The detailed interpretation of the experimental FRET results, however, relies on the comparison with structural modeling. We therefore present a complete first-principles modeling approach that explores the gas-phase structure of chromophore-grafted peptides and achieves accurate predictions of FRET efficiencies. We apply the approach to amyloid-β 12-28 fragments, known to be involved in amyloid plaque formation connected to Alzheimer's disease. We sample structures of the peptides that are grafted with 5-carboxyrhodamine 575 (Rh575) and QSY-7 chromophores by means of replica-exchange molecular dynamics simulations upon an Amber-type forcefield parametrization as a function of the charge state. The generated ensembles provide chromophore-distance and -orientation distributions which are used with the spectral parameters of the Rh575/QSY-7 chromophores to model FRET-efficiencies for the systems. The theoretical values agree with the experimental average "action"-FRET efficiencies and motivate to use the herein reported parametrization, sampling, and FRET-modeling technique in future studies on the structural properties and aggregation-behavior of related systems.Alexander Kulesza, Steven Daly, Luke MacAleese, Rodolphe Antoine, Philippe Dugourd