Interferon-β is a β-helical cytokine that plays a critical role in modulating neuroinflammatory pathways. We measured the stability of IFN-β, associated with equilibrium, in terms of change in Gibbs free energy (ΔGD0) between the folded (N) and denatured (D) states in the native buffer (i.e., during the absence of strong chemical denaturants, Guanidinium chloride (GdmCl) and urea are known to destroy noncovalent interactions responsible for maintaining the folded state). We have deliberated on the denaturation of IFN-β at 25 °C and pH 7.5 using two different spectroscopic probes, namely, fluorescence emission maxima (λmax, nm) and circular dichroism (ellipticity, θ in millidegrees) at 222 nm. From these in vitro studies, we reached the following conclusions: (i) Both denaturants induce a single step (“N” to “D”) transition. (ii) Denaturation of IFN-β protein is irreversible. (iii) Protein stability in terms of ΔGD0 is very low (ΔGD0 = ± 3.12 kcal·mol–1), which is likely due to the presence of hydrophobic patches (nonpolar side chains) on the surface of the native protein as shown by ANS (8-anilinonaphthalene-1-sulfonic acid) steady-state measurements. Atomistic simulations were conducted to obtain detailed molecular insights into the structural changes occurring with increased denaturant concentration (6 M GdmCl and 8 M urea) for a period of 500 ns in water at different temperatures (300–500 K). The analysis of results obtained from simulations corroborates those obtained from in vitro measurements. This investigation is the first comprehensive analysis of the unfolding trajectories of IFN-β under different denaturants.
Aiman et al. (Thu,) studied this question.