Quantitative analysis of the interaction strength and dynamics of human IgG4 half molecules by native mass spectrometry

Structure. 2011 Sep 7;19(9):1274-82. doi: 10.1016/j.str.2011.06.016.

Abstract

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 (IgG4Δhinge). 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Binding Sites, Antibody*
  • Computer Simulation
  • Humans
  • Immunoglobulin Fragments / chemistry*
  • Immunoglobulin Fragments / genetics
  • Immunoglobulin G / chemistry*
  • Immunoglobulin G / genetics
  • Kinetics
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Mutation, Missense
  • Protein Binding
  • Protein Stability
  • Spectrometry, Mass, Electrospray Ionization*
  • Thermodynamics

Substances

  • Immunoglobulin Fragments
  • Immunoglobulin G