63Cu(I) binding to human kidney 68Zn7-ßα MT1A: determination of Cu(I)-thiolate cluster domain specificity from ESI-MS and room temperature phosphorescence spectroscopy.

Mammalian metallothioneins (MTs) are important proteins in Zn(II) and Cu(I) homeostasis with the Zn(II) and Cu(I) binding to the 20 cysteines in metal-thiolate clusters. Previous electrospray ionization (ESI) mass spectrometric (MS) analyses of Cu(I) binding to Zn7-MT were complicated by significant overlap of the natural abundance isotopic patterns for Zn(II) and Cu(I) leading to impossibly ambiguous stoichiometries. In this paper, isotopically pure 63Cu(I) and 68Zn(II) allowed determination of the specific stoichiometries in the 68 Zn,63Cu-ßα MT1A species formed following the stepwise addition of 63Cu(I) to 68Zn7-ßα MT1A. These species were characterized by ESI-MS and room temperature emission spectroscopy. The key species that form and their emission band centres are Zn5Cu5-ßα MT1A (λ = 684 nm), Zn4Cu6-ßα MT1A (λ = 750 nm), Zn3Cu9-ßα MT1A (λ = 750 nm), Zn2Cu10-ßα MT1A (λ = 750 nm), and Zn1Cu14-ßα MT1A (λ = 634 nm). The specific domain stoichiometry of each species was determined by assessing the species forming following 63Cu(I) addition to the 68Zn3-ß MT1A and 68Zn4-α MT1A domain fragments. The domain fragment emission suggests that Zn5Cu5-ßα MT1A contains a Zn1Cu5-ß cluster and the Zn4Cu6-ßα MT1A, Zn3Cu9-ßα MT1A, and Zn2Cu10-ßα MT1A each contain a Cu6-ß cluster. The species forming with >10 mol. eq. of 63Cu(I) in ßα-MT1A exhibit emission from the Cu6-ß cluster and an α domain cluster. This high emission intensity is seen at the end of the titrations of 68Zn7-ßα MT1A and the 68Zn4-α MT1A domain fragment suggesting that the initial presence of the Zn(II) results in clustered Cu(I) binding in the α domain.

Formation of oxidative and non-oxidative dimers in metallothioneins: Implications for charge-state analysis for structural determination.

RATIONALE: Metallothioneins (MTs) are a class of dynamic proteins that have been investigated extensively using mass spectrometric methods due to their amenability to ionization. Here we detect the formation of oxidative and non-oxidative MT dimers using high-resolution mass spectrometry (HRMS) which has previously been overlooked with lower-resolution techniques. METHODS: Recombinant human MT1a and its isolated domain fragments were analyzed by high-resolution Thermo Q-Exactive and Bruker time-of-flight (TOF) mass spectrometers. Covalent Cys modification was performed using N-ethylmalemide to probe the effect of Cys oxidation on dimer formation. RESULTS: Dimerization was detected in the analysis of select charge states of Zn7 MT and apo-ßMT. Specifically, high resolution (140 k) revealed the +6 dimer peaks overlapping with the +3 charge state, but not with the other charge states (+4, +5, +6). The proteins with covalently modified Cys did not show dimer formation in any of their charge states. Apo-α and apo-ßαMT also did not form dimers under the conditions tested. CONCLUSIONS: Dimerization of MT was detected for zinc metalated and certain apo-MT forms with HRMS, which was not seen with lower-resolution techniques. These dimers appear overlapped only with certain charge states, confounding their analysis for structural characterization of MTs. The Zn-MT dimers appeared to be non-oxidative; however, the formation of dimers in the apo-protein is likely dependent on Cys oxidation.