TY - JOUR
T1 - Characterization of folding intermediates during urea-induced denaturation of human carbonic anhydrase II
AU - Wahiduzzaman,
AU - Dar, Mohammad Aasif
AU - Haque, Md Anzarul
AU - Idrees, Danish
AU - Hassan, Md Imtaiyaz
AU - Islam, Asimul
AU - Ahmad, Faizan
N1 - Funding Information:
WZ and MAD thanks to Indian Council of Medical Research (ICMR) for fellowships. MAH thanks the University Grants Commission (UGC) for fellowship. DI thanks Maulana Azad National Fellowship for the award of fellowship. This work was supported by grants from the Council of Scientific and Industrial Research (CSIR), India ( 37(1603)/13/EMR-II ) & ( 37(1604)/13/EMR-II ) and FIST Program ( SR/FST/LSI-541/2012 ). Authors are thankful to Dr. Abdul Waheed for providing gene of human carbonic anhydrase II.
Publisher Copyright:
© 2016 Elsevier B.V.
PY - 2017/2/1
Y1 - 2017/2/1
N2 - Knowledge of folding/unfolding pathway is fundamental basis to study protein structure and stability. Human carbonic anhydrase II (HCAII) is a ∼29 kDa, β-sheet dominated monomeric protein of 259 amino acid residues. In the present study, the urea-induced denaturation of HCAII was carried out which was a tri-phasic process, i.e., N (native) ↔ XI ↔ XII ↔ D (denatured) with stable intermediates XI and XII populated around 2 and 4 M urea, respectively. The far-UV CD was used to characterize the intermediate states (XI and XII) for secondary structural content, near-UV CD for tertiary structure, dynamic light scattering for hydrodynamic radius and ANS fluorescence spectroscopy for the presence of exposed hydrophobic patches. Based on these experiments, we concluded that urea-induced XI state has characteristics of molten globule state while XII state bears characteristics features of pre-molten globule state. Characterization of the intermediates on the folding pathway will contribute to a deeper understanding of the structure-function relationship of HCAII. Furthermore, this system may provide an excellent model to study urea stress and the strategies adopted by the organisms to combat such a stress.
AB - Knowledge of folding/unfolding pathway is fundamental basis to study protein structure and stability. Human carbonic anhydrase II (HCAII) is a ∼29 kDa, β-sheet dominated monomeric protein of 259 amino acid residues. In the present study, the urea-induced denaturation of HCAII was carried out which was a tri-phasic process, i.e., N (native) ↔ XI ↔ XII ↔ D (denatured) with stable intermediates XI and XII populated around 2 and 4 M urea, respectively. The far-UV CD was used to characterize the intermediate states (XI and XII) for secondary structural content, near-UV CD for tertiary structure, dynamic light scattering for hydrodynamic radius and ANS fluorescence spectroscopy for the presence of exposed hydrophobic patches. Based on these experiments, we concluded that urea-induced XI state has characteristics of molten globule state while XII state bears characteristics features of pre-molten globule state. Characterization of the intermediates on the folding pathway will contribute to a deeper understanding of the structure-function relationship of HCAII. Furthermore, this system may provide an excellent model to study urea stress and the strategies adopted by the organisms to combat such a stress.
KW - Human carbonic anhydrase II
KW - Molten globule state
KW - Protein folding
KW - Protein stability
KW - Urea-induced denaturation
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U2 - 10.1016/j.ijbiomac.2016.10.073
DO - 10.1016/j.ijbiomac.2016.10.073
M3 - Article
C2 - 27789330
AN - SCOPUS:85006000190
SN - 0141-8130
VL - 95
SP - 881
EP - 887
JO - International Journal of Biological Macromolecules
JF - International Journal of Biological Macromolecules
ER -