title: Structural basis of urea-induced unfolding: Unraveling the folding pathway of hemochromatosis factor E.
abstract: 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 222nm (θ222) versus [Urea], the molar urea concentration) revealed a biphasic transition with midpoint (Cm) values at 2.88M and 4.95M urea. Whereas, absorption analysis shows one two-state transition centered at 2.96M. To estimate the protein stability, denaturation curves were analyzed for Gibbs free energy change in the absence of urea (ΔGD(0)) 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 60ns was also performed. A clear correspondence was established between the in vitro and in silico studies.
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India.
authors.forename: Parvez
authors.initials: P
authors.lastname: Khan
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India.
authors.forename: Amresh
authors.initials: A
authors.lastname: Prakash
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India.
authors.forename: Md Anzarul
authors.initials: MA
authors.lastname: Haque
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India.
authors.forename: Asimul
authors.initials: A
authors.lastname: Islam
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India. Electronic address: mihassan@jmi.ac.in.
authors.forename: Md Imtaiyaz
authors.initials: MI
authors.lastname: Hassan
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India.
authors.forename: Faizan
authors.initials: F
authors.lastname: Ahmad
mesh_terms: D000071020:Hemochromatosis Protein / Q000737:chemistry*; D056004:Molecular Dynamics Simulation; D017433:Protein Structure, Secondary / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D058767:Protein Unfolding / Q000187:drug effects*; D014508:Urea / Q000494:pharmacology*
chemical_list: D000071020:Hemochromatosis Protein; D014508:Urea
keywords: Folding intermediate; Hemochromatosis; Pre-molten globule; Protein stability; Urea denaturation