Title
GdnHCl-induced unfolding intermediate in mitochondrial carbonic anhydrase VA
Author
Danish Idrees, Amresh Prakash, Md. Anzarul Haque, Asimul Islam, Md. Imtaiyaz Hassan, Faizan Ahmad
Year
2016
Journal
International Journal of Biological Macromolecules
Abstract
Carbonic anhydrase VA (CAVA) is a mitochondrial enzyme belonging to the α-family of CAs, which is involved in several physiological processes including ureagenesis, lipogenesis, gluconeogenesis and neuronal transmission. Here, we have tried to understand the folding mechanism of CAVA using guanidine hydrochloride (GdnHCl)-induced denaturation at pH 8.0 and 25 °C. The conformational stability was measured from the GdnHCl-induced denaturation study of CAVA monitored by circular dichroism (CD) and fluorescence measurements. On increasing the concentration of GdnHCl up to 5.0, a stable intermediate was observed between the concentrations 3.25 M to 3.40 M of the denaturant. However, CAVA gets completely denatured at 4.0 M GdnHCl. The existence of a stable intermediate state was validated by 1-anilinonaphthalene-8-sulfonic acid (ANS binding) fluorescence and near-UV CD measurements. In silico studies were also performed to analyse the effect of GdnHCl on the structure and stability of CAVA under explicit conditions. Molecular dynamics simulations for 40 ns were carried out and a well-defined correlation was established for both in vitro and in silico studies.
Instrument
J-1500
Keywords
Circular dichroism, Secondary structure, Tertiary structure, Protein denaturation, Thermodynamics, Biochemistry