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

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 ([θ]), Trp fluorescence emission at 342 nm (F) and difference molar absorption coefficient at 287 nm (Δε) were used to estimate stability parameters, [Formula: see text] (Gibbs free energy change in the absence of GdmCl; C (midpoint of the denaturation curve), i.e. molar GdmCl concentration ([GdmCl]) at which ΔG = 0; and m, the slope (=∂ΔG/∂[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 in silico and in vitro studies.