title: Spectroscopic and MD simulation studies on unfolding processes of mitochondrial carbonic anhydrase VA induced by urea.
abstract: Carbonic anhydrase VA (CAVA) is primarily expressed in the mitochondria and involved in numerous physiological processes including lipogenesis, insulin secretion from pancreatic cells, ureagenesis, gluconeogenesis and neuronal transmission. To understand the biophysical properties of CAVA, we carried out a reversible urea-induced isothermal denaturation at pH 7.0 and 25°C. Spectroscopic probes, [θ]222 (mean residue ellipticity at 222 nm), F344 (Trp-fluorescence emission intensity at 344 nm) and Δε280 (difference absorption at 280 nm) were used to monitor the effect of urea on the structure and stability of CAVA. The urea-induced reversible denaturation curves were used to estimate [Formula: see text], Gibbs free energy in the absence of urea; Cm, the mid-point of the denaturation curve, i.e. molar urea concentration ([urea]) at which ΔGD = 0; and m, the slope (=∂ΔGD/∂[urea]). Coincidence of normalized transition curves of all optical properties suggests that unfolding/refolding of CAVA is a two-state process. We further performed 40 ns molecular dynamics simulation of CAVA to see the dynamics at different urea concentrations. An excellent agreement was observed between in silico and in vitro studies.
authors.affiliation: a Centre for Interdisciplinary Research in Basic Sciences , Jamia Millia Islamia , Jamia Nagar, New Delhi 110025 , India.
authors.forename: Danish
authors.initials: D
authors.lastname: Idrees
authors.affiliation: a Centre for Interdisciplinary Research in Basic Sciences , Jamia Millia Islamia , Jamia Nagar, New Delhi 110025 , India.
authors.forename: Amresh
authors.initials: A
authors.lastname: Prakash
authors.affiliation: a Centre for Interdisciplinary Research in Basic Sciences , Jamia Millia Islamia , Jamia Nagar, New Delhi 110025 , India.
authors.forename: Md Anzarul
authors.initials: MA
authors.lastname: Haque
authors.affiliation: a Centre for Interdisciplinary Research in Basic Sciences , Jamia Millia Islamia , Jamia Nagar, New Delhi 110025 , India.
authors.forename: Asimul
authors.initials: A
authors.lastname: Islam
authors.affiliation: a Centre for Interdisciplinary Research in Basic Sciences , Jamia Millia Islamia , Jamia Nagar, New Delhi 110025 , India.
authors.forename: Faizan
authors.initials: F
authors.lastname: Ahmad
authors.affiliation: a Centre for Interdisciplinary Research in Basic Sciences , Jamia Millia Islamia , Jamia Nagar, New Delhi 110025 , India.
authors.forename: Md Imtaiyaz
authors.initials: MI
authors.lastname: Hassan
mesh_terms: D002256:Carbonic Anhydrases / Q000737:chemistry*; D002942:Circular Dichroism; D006801:Humans; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D058767:Protein Unfolding / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry* / Q000494:pharmacology
chemical_list: D014508:Urea; D002256:Carbonic Anhydrases
keywords: carbonic anhydrase VA; isothermal denaturation; molecular dynamics simulation; protein folding; protein stability; two-state unfolding; urea-induced denaturation