Copper speciation in the α and β domains of recombinant human metallothionein by electrospray ionization mass spectrometry

• Published in: Journal of inorganic biochemistry 2002-01, Vol.88 (2), p.153-172
• Main Authors: Merrifield, Maureen E, Huang, Zuyun, Kille, Peter, Stillman, Martin J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Copper; Copper - chemistry; Humans; Hydrogen-Ion Concentration; Mass Spectrometry; Metallothionein - chemistry; Metallothionein - isolation & purification; Models, Molecular; Molecular Sequence Data; Molecular Weight; Protein Conformation; Recombinant human metallothionein; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Spectrometry, Mass, Electrospray Ionization - methods; α domains; β domains
• ISSN: 0162-0134; 1873-3344
• DOI: 10.1016/S0162-0134(01)00394-4

ESI–MS data are reported for Cu(I) binding to the metal-free and cadmium-α and β domains of recombinant human metallothionein. These data provide information on the stoichiometric ratios of copper and cadmium that bind to the 11 thiolate sulfurs in the α fragment and the nine thiolate sulfurs in the β fragment. The data show the effects of the existing three-dimensional structure on the formation of different Cu(I)–thiolate clusters. Charge-state spectra are reported for a range of Cu(I) binding at low and neutral pH to the isolated α and β domains. There is an uneven distribution of charge states that show that changes in the three-dimensional structure take place as a function of Cu(I) loading. Metallation of the α domain at low pH takes place in a series of steps with the Cu 7 species dominating until at higher levels of Cu(I) the clusters become unstable resulting in increased concentrations of the metal-free being detected. We interpret this behavior as being the result of the expansion of the Cu–S domain structure to accommodate digonal co-ordination for the increased Cu(I) loading. This larger structure is unstable in the mass spectrometer and demetallation takes place. Metallation of the β domain at low pH proceeds in steps that involve initial formation of a Cu 5S 9 cluster, followed by the Cu 6S 9 at higher concentrations of Cu(I). The charge state spectra indicate a significant change in exposure of protonatable amino acids between Cu 5S 9 and Cu 6S 9 clusters, which indicates a change in peptide conformation when the Cu 6S 9 cluster forms. Metallation at neutral pH follows this same trend, namely, a much greater range of copper species is found during titrations of the Cd 4S 11-α fragment compared with the number of species that form when Cu(I) is added to Cd 3S 9-β. The mass spectral data indicate that at neutral pH, the presence of the tetrahedral geometry of the Cd(II) facilitates formation of mixed trigonal and digonal geometries for the incoming Cu(I) so that the most prominent species in the β fragment is Cd 1Cu 5S 9 which transforms into Cu 7S 9 at higher concentrations of Cu(I), and finally to Cu 9S 9 at saturation, all species involving a number of Cu(I) in digonal geometries. The observation that the metallation patterns of the α and β clusters follow different pathways at both low and neutral pH’s, suggests that the structures in the two domains are quite different, in agreement with previous proposals [J. Chem. Soc., Dalton Trans. (