Abstract
The presence of Cu, a highly redox active metal, is known to damage DNA as well as other cellular components, but the adverse effects of cellular Cu can be mitigated by metallothioneins (MT), small cysteine rich proteins that are known to bind to a broad range of metal ions. While metal ion binding has been shown to involve the cysteine thiol groups, the specific ion binding sites are controversial as are the overall structure and stability of the Cu-MT complexes. Here, we report results obtained using nano-electrospray ionization mass spectrometry and ion mobility-mass spectrometry for several Cu-MT complexes and compare our results with those previously reported for Ag-MT complexes. The data include determination of the stoichiometries of the complex (Cu(i)-MT, i = 1-19), and Cu(+) ion binding sites for complexes where i = 4, 6, and 10 using bottom-up and top-down proteomics. The results show that Cu(+) ions first bind to the β-domain to form Cu(4)MT then Cu(6)MT, followed by addition of four Cu(+) ions to the α-domain to form a Cu(10)-MT complex. Stabilities of the Cu(i)-MT (i = 4, 6 and 10) obtained using collision-induced unfolding (CIU) are reported and compared with previously reported CIU data for Ag-MT complexes. We also compare CIU data for mixed metal complexes (Cu(i)Ag(j)-MT, where i + j = 4 and 6 and Cu(i)Cd(j), where i + j = 4 and 7). Lastly, higher order Cu(i)-MT complexes, where i = 11-19, were also detected at higher concentrations of Cu(+) ions, and the metalated product distributions observed are compared to previously reported results for Cu-MT-1A (Scheller et al., Metallomics, 2017, 9, 447-462).