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

METALLOMICS(2023)

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摘要
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 Zn 7-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 Cu-63(I) and Zn-68(II) allowed determination of the specific stoichiometries in the Zn-68,Cu-63-beta alpha MT1A species formed following the stepwise addition of Cu-63(I) to Zn-68 7-beta alpha 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-beta alpha MT1A (lambda= 684nm), Zn4Cu6-beta alpha MT1A (lambda= 750nm), Zn3Cu9-beta aMT1A (lambda = 750 nm), Zn2Cu10-beta alpha MT1A (lambda = 750nm), and Zn1Cu14- beta alpha MT1A (lambda= 634nm). Thespecific domainstoichiometry ofeach specieswas determinedby assessingthe speciesforming following Cu-63(I) addition to the Zn-68(3)-beta MT1A and Zn-68(4)-alpha MT1A domain fragments. The domain fragment emission suggests that Zn5Cu5- beta alpha MT1A contains a Zn1Cu5-beta cluster and the Zn4Cu6-beta alpha MT1A, Zn3Cu9-beta alpha MT1A, and Zn2Cu10-beta alpha MT1A each contain a Cu 6-beta cluster. The species forming with >10 mol. eq. of Cu-63(I) in beta alpha-MT1A exhibit emission from the Cu 6-beta cluster and an adomain cluster. This high emission intensity is seen at the end of the titrations of Zn-68(7)-beta alpha MT1A and the Zn-68(4)-alpha MT1A domain fragment suggesting that the initial presence of the Zn(II) results in clustered Cu(I) binding in the adomain. [Graphics] .
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关键词
copper homeostasis,electrospray ionization mass spectrometry,emission spectroscopy,metallothionein,metalloprotein,zinc homeostasis
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