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Published on: July 9, 2013
Fluorescence studies on denaturation and stability of recombinant human interferon-gamma
Petya Christova1, Kristina Todorova, Ilijana Timtcheva
1Institute of Organic Chemistry with Center of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev, Str., bl. 9, 1113 Sofia, Bulgaria.
Insights
Recombinant human interferon-gamma (hIFNgamma) stability was investigated using chemical denaturants. Guanidine chloride proved a more potent denaturant than urea, suggesting minimal electrostatic contributions to hIFNgamma stability.
Area of Science:
- Biochemistry
- Protein chemistry
- Spectroscopy
Background:
- Recombinant human interferon-gamma (hIFNgamma) is a crucial therapeutic protein.
- Understanding protein folding and stability is essential for protein engineering and therapeutic applications.
Purpose of the Study:
- To investigate the unfolding/folding transitions of hIFNgamma in urea and guanidine chloride solutions.
- To determine the contribution of electrostatic interactions to hIFNgamma stability.
- To identify key residues influencing hIFNgamma stability.
Main Methods:
- Fluorescence spectroscopy was employed to monitor unfolding transitions.
- Chemical denaturation using urea and guanidine chloride at varying concentrations and pH.
- Application of the Stern-Volmer equation with iodide and acrylamide quenchers.
Main Results:
- Guanidine chloride (C* = 1.1 M, ΔG0 = 13.4 kJ/mol) was a more effective denaturant than urea (C* = 2.8 M, ΔG0 = 11.7 kJ/mol) at pH 7.4.
- The similar ΔG0 values suggest insignificant electrostatic contributions to hIFNgamma stability.
- hIFNgamma remained native in the pH range of 4.8-9.5.
- A cluster of acidic groups near the tryptophan residue was identified, potentially involving Asp63.
Conclusions:
- The stability of hIFNgamma is primarily governed by non-electrostatic forces.
- Protonation of Asp63 likely induces conformational changes affecting hIFNgamma stability.
- These findings provide insights into the structural dynamics of hIFNgamma.
Abstract:
Unfolding/folding transitions of recombinant human interferon-gamma (hIFNgamma) in urea and guanidine chloride (Gn.HCl) solutions were studied by fluorescence spectroscopy. At pH 7.4 Gn.HCl was a much more efficient denaturant (midpoint of unfolding C* = 1.1 M and deltaG0 = 13.4 kJ/mol) than urea (C* = 2.8 M and deltaG0 = 11.7 kJ/mol). The close deltaG0 values indicate that the contribution of electrostatic interactions to the stability of hIFNgamma is insignificant. Both the pH dependence of the fluorescence intensity and the unfolding experiments in urea at variable pH showed that hIFNgamma remains native in the pH range of 4.8-9.5. Using two quenchers, iodide and acrylamide, and applying the Stern-Volmer equation, a cluster of acidic groups situated in close proximity to the single tryptophan residue was identified. Based on the denaturation experiments at different pH values and on our earlier calculations of the electrostatic interactions in hIFNgamma, we assume that the protonation of Asp63 causes conformational changes having a substantial impact on the stability of hIFNgamma.
