International Journal of Biological Macromolecules 91 (2016) 1051–1061 Contents lists available at ScienceDirect International Journal of Biological Macromolecules j ourna l h o mepa ge: www.elsevier.com/locate/ijbiomac 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 Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India a r t i c l e i n f o Article history: Received 10 March 2016 Received in revised form 16 June 2016 Accepted 17 June 2016 Available online 23 June 2016 Keywords: Pre-molten globule Hemochromatosis Urea denaturation Protein stability Folding intermediate a b s t r a c t 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 (F 346 ) and CD signal at 222 nm ( 222 ) 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 (G 0 D ) associated with the equilibrium of denaturation exist between native state ↔ denatured state. The inter- mediate 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. © 2016 Elsevier B.V. All rights reserved. 1. Introduction The stability of the native state of globular protein is quite important, as this stability is marginal and constrained to a relatively fine gap of thermodynamic and solution composition conditions [1,2]. The progress in a deep understanding of the nature and extent of forces that tip the scales connecting the native and denatured states in terms of the individual roles of non- covalent interactions, entropy, temperature and pressure would have an impact on our capability to investigate native structures and abnormal aggregated states [3–5]. This further would help in the development of methods which will mimic biochemical pro- cesses. Deciphering how unfolding occurs, in terms of the dynamic pathway by which the unfolded state is recognized, is even more encouraging as it may give insight into the landscape that governs protein folding [6]. To fulfil the criteria of denaturation, till date a number of chemical denaturants have been used as an important and effi- ∗ Corresponding author. E-mail addresses: mihassan@jmi.ac.in, imtiyaz.hassan@gmail.com (Md.I. Hassan). cient method to disrupt protein structure to study successively the reverse process of folding [7,8]. Urea and guanidinium chlo- ride (GdmCl) are the two important denaturing agents which are largely used for folding-unfolding experiment [7,9,10]. However, this experiment unearthed a fundamental question, whether one can explain a denaturation process simply, independent of the denaturing agent. Privalov et al. [11,12] gave a clear answer to this issue, they conclude that the thermodynamic properties coupled with protein unfolding may not depend on the kind of denaturing agent. Levinthal [13] suggested that achievement of a native confor- mation of a protein should not be a random search; it must be regulated by well-defined intermediate states. Hence, the study of folding intermediates help us to understand the mechanism of pro- tein folding [14]. These protein folding intermediates are also very important for physiological functions such as transport across the membranes, respiration, cell-signalling or post-translational modi- fication, etc. [15–19]. Role of folding intermediates in many diseases has been also established [17,20]. Due to the immense importance of folding intermediates in protein folding and protein folding dis- eases, we must have a deep knowledge regarding the stability and structure of folding intermediates. Taking all the above informa- http://dx.doi.org/10.1016/j.ijbiomac.2016.06.055 0141-8130/© 2016 Elsevier B.V. All rights reserved.