Characterization of folding intermediates during urea-induced denaturation of human carbonic anhydrase II

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• 作者：Wahiduzzaman ; Mohammad Aasif Dar ; Md. Anzarul Haque ; Danish Idrees ; Md. Imtaiyaz Hassan ; Asimul Islam ; ^{aislam@jmi.ac.in} ; ^{asimulislams@gmail.com} ; Faizan Ahmad
• 关键词：Human carbonic anhydrase II ; Urea-induced denaturation ; Protein stability ; Protein folding ; Molten globule state
• 刊名：International Journal of Biological Macromolecules
• 出版年：2017
• 出版时间：February 2017
• 年：2017
• 卷：95
• 期：Complete
• 页码：881-887
• 全文大小：1045 K
• 卷排序：95

文摘

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.