Quantitative Analysis of the Interaction Strength and Dynamics of Human IgG4 Half Molecules by Native Mass Spectrometry
| Autor: | Stefan Loverix, Ignace Lasters, Janine Schuurman, Rebecca J. Rose, Aran F. Labrijn, Albert J. R. Heck, Paul W. H. I. Parren, Patrick Van Berkel, Ewald T. J. van den Bremer, Jan G. J. van de Winkel |
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| Přispěvatelé: | University of Groningen |
| Rok vydání: | 2011 |
| Předmět: |
Models
Molecular Spectrometry Mass Electrospray Ionization Dimer Electrospray ionization Amino Acid Motifs Kinetics Mutation Missense Analytical chemistry ANTIBODY C(H)3 DOMAIN Mass spectrometry VALIDATION CLASSIFICATION chemistry.chemical_compound FAB-ARM EXCHANGE Structural Biology parasitic diseases CIRCULAR-DICHROISM SPECTRA BINDING Humans Molecule Non-covalent interactions Computer Simulation DISULFIDE BONDS DIFFERENTIAL ALKYLATION Immunoglobulin Fragments Molecular Biology chemistry.chemical_classification REFINEMENT Protein Stability REDUCTION Crystallography Monomer chemistry Immunoglobulin G Mutagenesis Site-Directed Thermodynamics Binding Sites Antibody Quantitative analysis (chemistry) Protein Binding |
| Zdroj: | Structure, 19(9), 1274-1282. CELL PRESS |
| ISSN: | 0969-2126 |
| Popis: | Native mass spectrometry (MS) is a powerful technique for studying noncovalent protein-protein interactions. Here, native MS was employed to examine the noncovalent interactions involved in homodimerization of antibody half molecules (HL) in hinge-deleted human IgG4 (IgG4 Delta hinge). By analyzing the concentration dependence of the relative distribution of monomer HL and dimer (HL)(2) species, the apparent dissociation constant (K(D)) for this interaction was determined. In combination with site-directed mutagenesis, the relative contributions of residues at the CH3-CH3 interface to this interaction could be characterized and corresponding K(D) values quantified over a range of 10(-10)-10(-4) M. The critical importance of this noncovalent interaction in maintaining the intact dimeric structure was also proven for the full-length IgG4 backbone. Using time-resolved MS, the kinetics of the interaction could be measured, reflecting the dynamics of IgG4 HL exchange. Hence, native MS has provided a quantitative view of local structural features that define biological properties of IgG4. |
| Databáze: | OpenAIRE |
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