TY - JOUR
T1 - Structural basis of urea-induced unfolding
T2 - Unraveling the folding pathway of hemochromatosis factor E
AU - Khan, Parvez
AU - Prakash, Amresh
AU - Haque, Md Anzarul
AU - Islam, Asimul
AU - Hassan, Md Imtaiyaz
AU - Ahmad, Faizan
N1 - Funding Information:
PK thanks the Indian Council of Medical Research, India for the award of fellowship. MIH and FA thank to the Department of Science and Technology and India and Indian Council of Medical Research for financial support. AP and MAH are thankful to University Grants Commission (UGC) , New Delhi, India, for the award of fellowship. Authors thank Harvard University-PlasmID providing facility for providing the HFE gene. Suggestions from Dr. Abdul Waheed, St. Louis University are highly acknowledged. Authors sincerely thank the Department of Science and Technology, Government of India for the FIST support (FIST program No. SR/FST/LSI-541/2012).
Publisher Copyright:
© 2016 Elsevier B.V.
PY - 2016/10/1
Y1 - 2016/10/1
N2 - 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.
AB - 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.
KW - Folding intermediate
KW - Hemochromatosis
KW - Pre-molten globule
KW - Protein stability
KW - Urea denaturation
UR - http://www.scopus.com/inward/record.url?scp=84977268048&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84977268048&partnerID=8YFLogxK
U2 - 10.1016/j.ijbiomac.2016.06.055
DO - 10.1016/j.ijbiomac.2016.06.055
M3 - Article
C2 - 27339324
AN - SCOPUS:84977268048
SN - 0141-8130
VL - 91
SP - 1051
EP - 1061
JO - International Journal of Biological Macromolecules
JF - International Journal of Biological Macromolecules
ER -