Collision-Induced Unfolding of Partially Metalated Metallothionein-2A: Tracking Unfolding Reactions of Gas-Phase Ions

Abstract

Metallothioneins (MTs) constitute a group of intrinsically disordered proteins that exhibit extreme diversity in structure, biological functionality, and metal ion specificity. Structures of coordinatively saturated metalated MTs have been extensively studied, but very limited structural information for the partially metalated MTs exists. Here, the conformational preferences from partial metalation of rabbit metallothionein-2A (MT) by Cd, Zn, and Ag are studied using nanoelectrospray ionization ion mobility mass spectrometry. We also employ collision-induced unfolding to probe differences in the gas-phase stabilities of these partially metalated MTs. Our results show that despite their similar ion mobility profiles, Cd-MT, Zn-MT, Ag-MT, and Ag-MT differ dramatically in their gas-phase stabilities. Furthermore, the sequential addition of each Cd and Zn ion results in the incremental stabilization of unique unfolding intermediates.

Compaction of RNA Hairpins and Their Kissing Complexes in Native Electrospray Mass Spectrometry

“…This recalls the offset collision-induced unfolding of proteins in presence of divalent cations. [45][46] To summarize, together with our previous study on DNA and RNA double helices, 11 our results…”

Metal- and Affinity-Specific Dual Labeling of Cysteine-Rich Proteins for Identification of Metal-Binding Sites

“…Recently, a paper reported collision-induced unfolding experiments followed through ion mobility-MS for Zn 4 MT2. 74 The authors reported four separate conformers with regard to collision cross section (CCS) profiles that required a different collision activation for unfolding. The β-domain is not fully extended but rather folded for Cd 4 MT2, as opposed to Zn 4 MT2, which showed a CCS of around 1000, which corresponds to the β-domain being fully extended.…”

“…However, this idea has been supported by neither Russell’s group nor here. 74 , 75 Russell’s group concluded that the region from Asn18 to Cys38 participates in the coordination of the first four Zn 2+ ions, but the terminal Cys5 or Cys7 are at most weakly interacting. Here, with the experimental conditions employed, we suggest that the region 21–30 participates in Zn 2+ binding.…”

“…Despite the above differences, our results agreed and suggested that there exists a Zn 2+ redistribution between both α- and β-domains. 74 , 75 …”

Metallomics: Methodologies and Applications in Environmental Studies

“…Shen et al 105 e Ngu et al 106 investigaram metalotioneínas associadas ao Cu e Cd por ESI-MS, o uso de ICP-MS em combinação com a técnica de separação mostrou-se eficiente na análise de ferramentas das isoformas de MTs. 9,52 Dong et al 19 utilizando uma combinação de mobilidade iônica com nESI-MS, foram capazes de determinar diretamente as várias conformações que a população iônica pode apresentar, bem como as informações de tamanho de íons que podem estar relacionadas à estrutura e conformação.…”