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The melting of native domain structure in effector activation of IgG studied by using congo red as a specific probe
B Piekarska1, I Roterman, J Rybarska
1Institute of Medical Biochemistry, Collegium Medicum, Jagiellonian University, Cracow, Poland.
Insights
Structural changes in immunoglobulin G (IgG) molecules, induced by antigen binding or heat, reveal a molten globule state. This destabilization enhances susceptibility to digestion and impacts immune complex stability and effector functions.
Area of Science:
- Immunology
- Structural Biology
- Biochemistry
Background:
- Immunoglobulin G (IgG) structure is crucial for its function.
- Understanding IgG conformational changes upon antigen binding or heat aggregation is vital for immune response mechanisms.
Purpose of the Study:
- To investigate structural alterations in IgG molecules upon antigen binding and heat aggregation.
- To characterize the conformation of IgG associated with Congo Red dye binding.
- To assess the impact of these structural changes on IgG susceptibility to proteolysis and effector functions.
Main Methods:
- Utilized bis azo dye (Congo Red) as a specific probe to study IgG structural changes.
- Analyzed dye-protein complex properties to infer peptide chain unfolding and packing.
- Assessed susceptibility of modified IgG to enzymatic digestion.
- Performed energetic evaluation of IgG domain stability.
Main Results:
- IgG conformation responsible for dye binding exhibits characteristics of an unfolding intermediate, akin to a molten globule state.
- Dye-protein complexes show signs of peptide chain unfolding with preserved compact packing.
- Heat- or antigen-induced IgG becomes more susceptible to digestion, yielding a 30,000 D peptide fragment likely from the CH1 domain of the heavy chain.
- Energetic analysis identifies the CH1 domain as the least stable IgG fragment.
Conclusions:
- Destabilized tertiary packing in antigen-bound IgG may promote association of antibody molecules.
- This association can enhance immune complex stability and influence effector activity.
- The CH1 domain's conformational lability plays a key role in IgG structural dynamics and function.
Abstract:
The nature of structural changes in IgG molecules associated with the binding to antigen and/or heat aggregation was studied using bis azo dye (Congo Red) as the specific probe. It was found, that protein conformation responsible for binding the dye represents an unfolding intermediate with properties corresponding to a molten globule state. The properties of the dye-protein complex reveal the signs of an unfolding of the peptide chain with simultaneously preserved relatively compact packing. Immunoglobulins which were induced by heating, or binding to antigen in order to form the complex with dye ligands, become more susceptible for digestion. The main peptide of molecular weight 30,000 D which appears in products was suggested to originate from a heavy chain after its splitting in the region of CH1 domain. The energetic evaluation of stability of IgG domains also indicates that CH1 is the least stable fragment of the heavy chain and its conformation may be destabilized first. It was concluded that destabilized tertiary packing of antibodies bound to antigen may favour the association of closely situated immunoglobulin molecules increasing the stability of the immune complex and influencing in the result its effector activity.
