GdmCl-induced unfolding studies of human carbonic anhydrase IX: a combined spectroscopic and MD simulation approach

Abstract

<p>Carbonic anhydrase IX (CAIX) is a transmembrane glycoprotein, associated with tumor, acidification which leads to the cancer, and is considered as a potential biomarker for hypoxia-induced cancers. The overexpression of CAIX is linked with hypoxia condition which is mediated by the transcription of hypoxia-induced factor (HIF-1). To understand the biophysical properties of CAIX, we have carried out a reversible isothermal denaturation of CAIX-induced by GdmCl at pH 8.0 and 25°C. Three different spectroscopic probes, the far-UV CD at 222 nm ([<i>θ</i>]₂₂₂), Trp fluorescence emission at 342 nm (<i>F</i>₃₄₂) and difference molar absorption coefficient at 287 nm (Δ<i>ε</i>₂₈₇) were used to estimate stability parameters, (Gibbs free energy change in the absence of GdmCl; <i>C</i>_<i>m</i> (midpoint of the denaturation curve), i.e. molar GdmCl concentration ([GdmCl]) at which Δ<i>G</i>_D = 0; and <i>m</i>, the slope (=∂Δ<i>G</i>_D/∂[GdmCl])). GdmCl induces a reversible denaturation of CAIX. Coincidence of the normalized transition curves of all optical properties suggests that unfolding/refolding of CAIX is a two-state process. We further performed molecular dynamics simulation of CAIX for 40 ns to see the dynamics of protein structure in different GdmCl concentrations. An excellent agreement was observed between <i>in silico</i> and <i>in vitro</i> studies.</p