Structural properties of metal-free apometallothioneins

Abstract

The metalated forms of metallothionein are well studied (particularly Zn–MT, Cu–MT and Cd–MT), but almost nothing is known about the chemical and structural properties of apometallothioneins despite their importance in initial metalation and subsequent demetalation. Electrospray ionization mass spectrometry was used to provide a detailed view of the structural properties of the metal-free protein. Mass spectra of Zn7–MT and apo-MT at pH 7 exhibit the same charge state distribution, indicating that apo-MT is tightly folded like the metallated protein, whereas apo-MT at pH 3 exhibits a charge state spectrum associated with unfolding or denaturation. Benzoquinone was used to modify the cysteines in the β-MT (9Bq), and α-MT (11Bq) fragments, and the full βα-MT (20Bq) protein. ESI-MS showed that the overall volume and, therefore, the extent of folding for the modified proteins is similar to that of Zn–MT. Molecular modeling using MM3-MD methods provided the volume of each modified protein. The volumes of the partially modified proteins follow the same trend as the charge states, showing that ESI-MS is an excellent method with which to follow small changes in protein folding as a function of applied chemical stress. The data suggest that the structure of apo-βα-MT is more organized than previously considered.