Measurement of dissociation rate of biomolecular complexes using CE.

Electrophoresis
Peilin YangRobert T Kennedy

Abstract

Fluorescence anisotropy (FA), non-equilibrium CE of equilibrium mixtures (NECEEM) and high-speed CE were evaluated for measuring dissociation kinetics of peptide-protein binding systems. Fyn-SH3-SH2, a protein construct consisting of the src homology 2 (SH2) and 3 (SH3) domain of the protein Fyn, and a fluorescein-labeled phosphopeptide were used as a model system. All three methods gave comparable half-life of approximately 53 s for Fyn-SH3-SH2:peptide complex. Achieving satisfactory results by NECEEM required columns over 30 cm long. When using Fyn-SH2-SH3 tagged with glutathione S-transferase (GST) as the binding protein, both FA and NECEEM assays gave evidence of two complexes forming with the peptide, yet neither method allowed accurate measurement of dissociation rates for both complexes because of a lack of resolution. High-speed CE, with a 7 s separation time, enabled separation of both complexes and allowed determination of dissociation rate of both complexes independently. The two complexes had half-lives of 22.0+/-2.7 and 58.8+/-6.1 s, respectively. Concentration studies revealed that the GST-Fyn-SH3-SH2 protein formed a dimer so that complexes had binding ratios of 2:1 (protein-to-peptide ratio) and 2:2. Our results...Continue Reading

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Citations

May 21, 2013·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Eunmi Ban, Eun Joo Song
Jul 7, 2011·Analytical Chemistry·Stephanie de Jong, Sergey N Krylov
Jan 27, 2011·Nature Communications·Andreas OttoDörte Becher
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Mar 1, 2012·The Analyst·Leonid T Cherney, Sergey N Krylov
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Apr 27, 2010·Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy·Gehad G Mohamed, Madiha H Soliman
Apr 5, 2016·Journal of the American Chemical Society·Friederike M MöllerDieter Braun
Jun 25, 2016·Analytical Chemistry·Mirzo Kanoatov, Sergey N Krylov
Dec 3, 2016·Expert Opinion on Drug Discovery·Claire M OuimetRobert T Kennedy
May 26, 2010·Analytical Chemistry·Nicholas W FrostMichael T Bowser
Aug 7, 2021·Electrophoresis·Anjali Gopal, Amy E Herr

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