1 research outputs found
Protein Cross-Linking Capillary Electrophoresis for Protein–Protein Interaction Analysis
Capillary
electrophoresis (CE) has been identified as a useful
platform for detecting, quantifying, and screening for modulators
of protein–protein interactions (PPIs). In this method, one
protein binding partner is labeled with a fluorophore, the protein
binding partners are mixed, and then, the complex is separated from
free protein to allow direct determination of bound to free ratios.
Although it possesses many advantages for PPI studies, the method
is limited by the need to have separation conditions that both prevent
protein adsorption to capillary and maintain protein interactions
during the separation. In this work, we use protein cross-linking
capillary electrophoresis (PXCE) to overcome this limitation. In PXCE,
the proteins are cross-linked under binding conditions and then separated.
This approach eliminates the need to maintain noncovalent interactions
during electrophoresis and facilitates method development. We report
PXCE methods for an antibody–antigen interaction and heterodimer
and homodimer heat shock protein complexes. Complexes are cross-linked
by short treatments with formaldehyde after reaching binding equilibrium.
Cross-linked complexes are separated by electrophoretic mobility using
free solution CE or by size using sieving electrophoresis of SDS complexes.
The method gives good quantitative results; e.g., a lysozyme–antibody
interaction was found to have <i>K</i><sub>d</sub> = 24
± 3 nM by PXCE and <i>K</i><sub>d</sub> = 17 ±
2 nM using isothermal calorimetry (ITC). Heat shock protein 70 (Hsp70)
in complex with bcl2 associated athanogene 3 (Bag3) was found to have <i>K</i><sub>d</sub> = 25 ± 5 nM by PXCE which agrees with <i>K</i><sub>d</sub> values reported without cross-linking. Hsp70–Bag3
binding site mutants and small molecule inhibitors of Hsp70–Bag3
were characterized by PXCE with good agreement to inhibitory constants
and IC<sub>50</sub> values obtained by a bead-based flow cytometry
protein interaction assay (FCPIA). PXCE allows rapid method development
for quantitative analysis of PPIs