Molecular Basis of the Structural Stability of Hemochromatosis Factor E: A Combined Molecular Dynamic Simulation and GdmCl-Induced Denaturation Study Parvez Khan, Amresh Parkash, Asimul Islam, Faizan Ahmad, Md. Imtaiyaz Hassan Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India Received 23 September 2015; revised 26 October 2015; accepted 30 October 2015 Published online 5 November 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22760 ABSTRACT: Hemochromatosis factor E (HFE) is a member of class I MHC family and plays a significant role in the iron homeostasis. Denaturation of HFE induced by guanidi- nium chloride (GdmCl) was measured by monitoring changes in [u] 222 (mean residue ellipticity at 222 nm), intrinsic fluorescence emission intensity at 346 nm (F 346 ) and the difference absorption coefficient at 287 nm (De 287 ) at pH 8.0 and 258C. Coincidence of denaturation curves of these optical properties suggests that GdmCl- induced denaturation (native (N) state $ denatured (D) state) is a two-state process. The GdmCl-induced denaturation was found reversible in the entire concen- tration range of the denaturant. All denaturation curves were analyzed for DG 0 D , Gibbs free energy change associ- ated with the denaturation equilibrium (N state $ D state) in the absence of GdmCl, which is a measure of HFE stability. We further performed molecular dynamics simulation for 40 ns to see the effect of GdmCl on the structural stability of HFE. A well defined correlation was established between in vitro and in silico studies. V C 2015 Wiley Periodicals, Inc. Biopolymers 105: 133–142, 2016. Keywords: GdmCl-induced denaturation; hemochroma- tosis factor E; protein stability; protein folding; molecu- lar dynamics simulation; two-state denaturation This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the pre- print version. You can request a copy of any preprints from the past two calendar years by emailing the Biopolymers edi- torial office at biopolymers@wiley.com. INTRODUCTION P roteins are essential biological macromolecules hav- ing immense importance in every physiochemical process. To perform their biological function in a regulated way, a protein must attains a well defined conformation, known as folded state or native state. 1 Most of the proteins in solution try to remain in the native or folded state which is governed by pH, ionic strength, temperature 2 and the presence and absence of other cofactors and proteins like chaperons. 3,4 Stability of proteins can also be controlled with the addition of osmolytes (pro- line, trehalose, trimethylamine N-oxide) which stabilize the native state. 5,6 On the other hand, guanidinium chloride (GdmCl) and urea are major denaturing agents for proteins. 7 Chemical denaturants have been used as an efficient method to disrupt all non-covalent interaction responsible for the stability of the native protein conformation. 8,9 A vast litera- ture exists on the determination of protein stability from the analysis of reversible a two-state denaturation curves. 10–16 Hemochromatosis factor E (HFE) is a transmembrane type class MHC I protein and plays an important role in iron uptake and regulation. 17,18 The role of HFE in iron homeosta- sis is implicated by the discovery that it is mutated in patients Additional Supporting Information may be found in the online version of this article. Correspondence to: Dr. Md. Imtaiyaz Hassan; e-mail: mihassan@jmi.ac.in Contract grant sponsor: Department of Science and Technology, Government of India, Contract grant number: FIST program No. SR/FST/LSI-541/2012 V C 2015 Wiley Periodicals, Inc. Biopolymers Volume 105 / Number 3 133