Human apo-metallothionein 1a is not a random coil: Evidence from guanidinium chloride, high temperature, and acidic pH unfolding studies

Abstract

The structures of apo-metallothioneins (apo-MTs) have been relatively elusive due to their fluxional, disordered state which has been difficult to characterize. However, intrinsically disordered protein (IDP) structures are rather diverse, which raises questions about where the structure of apo-MTs fit into the protein structural spectrum. In this paper, the unfolding transitions of apo-MT1a are discussed with respect to the effect of the chemical denaturant GdmCl, temperature conditions, and pH environment. Cysteine modification in combination with electrospray ionization mass spectrometry was used to probe the unfolding transition of apo-MT1a in terms of cysteine exposure. Circular dichroism spectroscopy was also used to monitor the change in secondary structure as a function of GdmCl concentration. For both of these techniques, cooperative unfolding was observed, suggesting that apo-MT1a is not a random coil. More GdmCl was required to unfold the protein backbone than to expose the cysteines, indicating that cysteine exposure is likely an early step in the unfolding of apo-MT1a. MD simulations complement the experimental results, suggesting that apo-MT1a adopts a more compact structure than expected for a random coil. Overall, these results provide further insight into the intrinsically disordered structure of apo-MT.