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.

Structural Characterization of Ectodomain G Protein of Respiratory Syncytial Virus and Its Interaction with Heparan Sulfate: Multi-Spectroscopic and In Silico Studies Elucidating Host-Pathogen Interactions

“…The protein stability can be measured by equilibrium unfolding studies in the presence of GdmCl or urea [ 48 ]. The edG has one tryptophan and five tyrosine residues which are often buried either partially or fully in the hydrophobic core of the folded protein.…”

Effect of pH on the structure, function, and stability of human calcium/calmodulin-dependent protein kinase IV: combined spectroscopic and MD simulation studies

“…MD simulation in combinations with experimental studies help to get better insight into the conformation of protein in different environments (Anwer et al 2015;Idrees et al 2015;Khan et al 2016a;Khan et al 2016b;Khan et al 2016c;Naiyer et al 2015;Ubaid-Ullah et al 2014). After equilibration CAMKIV was simulated for 20 ns at different protonation state, its behavior is analyzed using the utilities of GROMACS.…”

In silico and In vitro Perspective to Elucidate the Anticancer Effect of Natural Compound Hesperetin in Inhibiting Mammalian Target of Rapamycin Pathway

“…MD simulation has become an indispensable tool in computational biology research towards understanding the dynamic behavior of molecular complexes and intermolecular interactions in physiologically simulated conditions [30][31][32][33] . Hence, in this study, we implemented explicit MD simulations using the Desmond package (Desmond MD System, version 3.1, D. E. Shaw Research, New York, NY, 2012; Maestro-Desmond Interoperability Tools, version 3.1, Schrödinger, New York, NY, 2012) with OPLS2005 as force field.…”