Copper speciation in the alpha and beta domains of recombinant human metallothionein by electrospray ionization mass spectrometry.
ESI-MS data are reported for Cu(I) binding to the metal-free and cadmium-alpha and beta 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 alpha fragment and the nine thiolate sulfurs in the beta 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 alpha and beta 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 alpha domain at low pH takes place in a series of steps with the Cu7 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 beta domain at low pH proceeds in steps that involve initial formation of a Cu5S9 cluster, followed by the Cu6S9 at higher concentrations of Cu(I). The charge state spectra indicate a significant change in exposure of protonatable amino acids between Cu5S9 and Cu6S9 clusters, which indicates a change in peptide conformation when the Cu6S9 cluster forms. Metallation at neutral pH follows this same trend, namely, a much greater range of copper species is found during titrations of the Cd4S11-alpha fragment compared with the number of species that form when Cu(I) is added to Cd3S9-beta. 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 beta fragment is Cd1Cu5S9 which transforms into Cu7S9 at higher concentrations of Cu(I), and finally to Cu9S9 at saturation, all species involving a number of Cu(I) in digonal geometries. The observation that the metallation patterns of the alpha and beta 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
Merrifield
Maureen E
ME
Department of Chemistry, The University of Western Ontario, London, Ontario, Canada N6A 5B7.
Huang
Zuyun
Z
Kille
Peter
P
Stillman
Martin J
MJ
D000595:Amino Acid Sequence; D003300:Copper / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008668:Metallothionein / Q000737:chemistry* / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D011994:Recombinant Proteins; D003300:Copper; D008668:Metallothionein