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

Parvez Khan, Amresh Prakash, Md Anzarul Haque, Asimul Islam, Md Imtaiyaz Hassan, Faizan Ahmad

Research output: Contribution to journalArticlepeer-review


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 (Cm) 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 (ΔGD0) 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.

Original languageEnglish (US)
Pages (from-to)1051-1061
Number of pages11
JournalInternational Journal of Biological Macromolecules
StatePublished - Oct 1 2016
Externally publishedYes


  • Folding intermediate
  • Hemochromatosis
  • Pre-molten globule
  • Protein stability
  • Urea denaturation

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Economics and Econometrics
  • General Energy


Dive into the research topics of 'Structural basis of urea-induced unfolding: Unraveling the folding pathway of hemochromatosis factor E'. Together they form a unique fingerprint.

Cite this