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Structural features of interferon-gamma aggregation revealed by hydrogen exchange
Scott A Tobler1, Erik J Fernandez
1Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia 22904-4741, USA.
Summary
Investigating recombinant human interferon-gamma (IFN-gamma) aggregation using hydrogen-deuterium exchange (HX) revealed that helix C remains intact in aggregates. Native-state HX patterns may predict protein regions prone to unfolding during aggregation.
Area of Science:
- Biochemistry
- Protein Chemistry
- Structural Biology
Background:
- Recombinant human interferon-gamma (IFN-gamma) is a crucial therapeutic protein.
- Protein aggregation is a significant challenge in biopharmaceutical development, impacting protein stability and efficacy.
- Understanding the structural basis of protein aggregation is essential for developing stable therapeutic proteins.
Purpose of the Study:
- To investigate the stability and structural changes of recombinant human interferon-gamma (IFN-gamma) during aggregation induced by guanidine hydrochloride (GdnHCl) and potassium thiocyanate (KSCN).
- To probe the tertiary structure of aggregated IFN-gamma and assess its stability under aggregation-prone conditions.
- To identify specific regions of IFN-gamma that are susceptible to unfolding during aggregation.
Main Methods:
- Hydrogen-deuterium exchange (HX) coupled with electrospray ionization mass spectrometry (ESI-MS).
- HX labeling was performed on both pre-formed amorphous aggregates and at low protein concentrations to assess stability.
- Comparative analysis of HX patterns under native, partially unfolded, and aggregated states induced by GdnHCl and KSCN.
Main Results:
- In 1 M GdnHCl, IFN-gamma stability was reduced, with less protection from HX, though helix C showed slower exchange, indicating partial unfolding with intact helix C in aggregates.
- Aggregates formed in 0.3 M KSCN showed similar HX patterns to GdnHCl-induced aggregates, but solution phase HX in KSCN resembled the native state.
- Potassium thiocyanate initially precipitated native protein before facilitating partial unfolding, while helix C remained intact in both GdnHCl- and KSCN-induced aggregates.
- Helix C, the hydrophobic core of the IFN-gamma dimer, demonstrated high protection under native conditions and greater stability in GdnHCl.
Conclusions:
- Helix C of IFN-gamma remains structurally intact during aggregation induced by both GdnHCl and KSCN.
- Native-state HX patterns can potentially predict regions of IFN-gamma susceptible to unfolding during the aggregation process.
- These findings provide insights into the structural mechanisms of IFN-gamma aggregation, aiding in the development of more stable therapeutic formulations.