title: GdnHCl-induced unfolding intermediate in the mitochondrial carbonic anhydrase VA.
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 3.25M to 3.40M of the denaturant. However, CAVA gets completely denatured at 4.0M 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 40ns were carried out and a well-defined correlation was established for both in vitro and in silico studies.
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, 110025, India.
authors.forename: Danish
authors.initials: D
authors.lastname: Idrees
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, 110025, India.
authors.forename: Amresh
authors.initials: A
authors.lastname: Prakash
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, 110025, India.
authors.forename: Md Anzarul
authors.initials: MA
authors.lastname: Haque
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, 110025, India.
authors.forename: Asimul
authors.initials: A
authors.lastname: Islam
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, 110025, India. Electronic address: mihassan@jmi.ac.in.
authors.forename: Md Imtaiyaz
authors.initials: MI
authors.lastname: Hassan
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, 110025, India.
authors.forename: Faizan
authors.initials: F
authors.lastname: Ahmad
mesh_terms: D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008928:Mitochondria / Q000201:enzymology*; D056004:Molecular Dynamics Simulation; D011489:Protein Denaturation / Q000187:drug effects*; D013696:Temperature
chemical_list: D002256:Carbonic Anhydrases; D019791:Guanidine
keywords: 1-Anilinonaphthalene-8-sulfonic acid; Carbonic anhydrase VA; GdnHCl-induced denaturation; Molecular dynamics simulation; Protein folding