Title
Characterization of folding intermediates during urea-induced denaturation of human carbonic anhydrase II
Author
Wahiduzzaman, Mohammad Aasif Dar, Md. Anzarul Haque, Danish Idrees, Md. Imtaiyaz Hassan, Asimul Islam, Faizan Ahmad
Year
2016
Journal
International Journal of Biological Macromolecules
Abstract
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 (XIand 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.
Instrument
FP-6200
Keywords
Fluorescence, Protein structure, Protein folding, Biochemistry