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

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 ] 222 ), Trp fluorescence emission at 342 nm ( F 342 ) and difference molar absorption coefficient at 287 nm (Δ ε 287 ) were used to estimate stability parameters, (Gibbs free energy change in the absence of GdmCl; C m (midpoint of the denaturation curve), i.e. molar GdmCl concentration ([GdmCl]) at which Δ G D = 0; and m , the slope (=∂Δ G 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.