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Measurement of dissociation rate of biomolecular complexes using CE
Author(s) -
Yang Peilin,
Mao Yingwei,
Lee Angel W.M.,
Kennedy Robert T.
Publication year - 2009
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200800397
Subject(s) - phosphopeptide , dimer , chemistry , fluorescence anisotropy , peptide , sh3 domain , dissociation (chemistry) , kinetics , fyn , sh2 domain , receptor–ligand kinetics , crystallography , biophysics , chromatography , proto oncogene tyrosine protein kinase src , biochemistry , phosphorylation , biology , organic chemistry , physics , receptor , quantum mechanics , membrane
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∼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 demonstrate that although all methods are suitable for 1:1 binding systems, high‐speed CE is unique in allowing multiple complexes to be resolved simultaneously. This property allows determination of binding kinetics of complicated systems and makes the technique useful for discovering novel affinity interactions.