title: Protein Cross-Linking Capillary Electrophoresis for Protein-Protein Interaction Analysis.
abstract: 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 Kd = 24 ± 3 nM by PXCE and Kd = 17 ± 2 nM using isothermal calorimetry (ITC). Heat shock protein 70 (Hsp70) in complex with bcl2 associated athanogene 3 (Bag3) was found to have Kd = 25 ± 5 nM by PXCE which agrees with Kd 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 IC50 values obtained by a bead-based flow cytometry protein interaction assay (FCPIA). PXCE allows rapid method development for quantitative analysis of PPIs.
authors.affiliation: Department of Chemistry, University of Michigan , 930 N. University Ave., Ann Arbor, Michigan 48109, United States.
authors.forename: Claire M
authors.initials: CM
authors.lastname: Ouimet
authors.affiliation: Department of Pharmaceutical Chemistry and the Institute for Neurodegenerative Disease, University of California at San Francisco , 675 Nelson Rising Ln., San Francisco, California 94158, United States.
authors.forename: Hao
authors.initials: H
authors.lastname: Shao
authors.affiliation: Department of Pharmaceutical Chemistry and the Institute for Neurodegenerative Disease, University of California at San Francisco , 675 Nelson Rising Ln., San Francisco, California 94158, United States.
authors.forename: Jennifer N
authors.initials: JN
authors.lastname: Rauch
authors.affiliation: Department of Chemistry, University of Michigan , 930 N. University Ave., Ann Arbor, Michigan 48109, United States.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Dawod
authors.affiliation: Department of Molecular Medicine, University of South Florida , 4001 E. Fletcher Ave., MDC 36, Tampa, Florida 33613, United States.
authors.forename: Bryce
authors.initials: B
authors.lastname: Nordhues
authors.affiliation: Department of Molecular Medicine, University of South Florida , 4001 E. Fletcher Ave., MDC 36, Tampa, Florida 33613, United States.
authors.forename: Chad A
authors.initials: CA
authors.lastname: Dickey
authors.affiliation: Department of Pharmaceutical Chemistry and the Institute for Neurodegenerative Disease, University of California at San Francisco , 675 Nelson Rising Ln., San Francisco, California 94158, United States.
authors.forename: Jason E
authors.initials: JE
authors.lastname: Gestwicki
authors.affiliation: Department of Chemistry, University of Michigan , 930 N. University Ave., Ann Arbor, Michigan 48109, United States.|Department of Pharmacology, University of Michigan , 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109, United States.
authors.forename: Robert T
authors.initials: RT
authors.lastname: Kennedy
mesh_terms: D000937:Antigen-Antibody Reactions / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002151:Calorimetry; D003432:Cross-Linking Reagents / Q000737:chemistry*; D019281:Dimerization; D019075:Electrophoresis, Capillary; D005456:Fluorescent Dyes / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding
chemical_list: D051017:Apoptosis Regulatory Proteins; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D006360:Heat-Shock Proteins; D005557:Formaldehyde; D009113:Muramidase