Structural basis of urea-induced unfolding: Unraveling the folding pathway of hemochromatosis factor E

Hereditary hemochromatosis factor E (HFE) is a type 1 transmembrane protein, and acts as a negative regulator of iron-uptake. The equilibrium unfolding and conformational stability of the HFE protein was examined in the presence of urea. The folding and unfolding transitions were monitored with the help of circular dichroism (CD), intrinsic fluorescence and absorption spectroscopy. Analysis of transition curves revealed that the folding of HFE is not a two-state process. However, it involved stable intermediates. Transition curves (plot of fluorescence (F₃₄₆) and CD signal at 222 nm (θ₂₂₂) versus [Urea], the molar urea concentration) revealed a biphasic transition with midpoint (C_m) values at 2.88 M and 4.95 M urea. Whereas, absorption analysis shows one two-state transition centered at 2.96 M. To estimate the protein stability, denaturation curves were analyzed for Gibbs free energy change in the absence of urea (ΔG_D⁰) associated with the equilibrium of denaturation exist between native state ↔ denatured state. The intermediate state was further characterized by hydrophobic probe, 1-anilinonaphthalene-8-sulfonic acid (ANS-binding). For seeing the effect of urea on the structure and dynamics of HFE, molecular dynamics simulation for 60 ns was also performed. A clear correspondence was established between the in vitro and in silico studies.