| dc.description.abstract | Metallothioneins (MTs) constitute a family of cysteine-rich proteins that have the ability to bind a wide range of metal ions including Cd^2+, Zn^2+ and Ag^+ . MTs are important in metal homeostasis and detoxification. Even through apo- and partially metalated MTs are physiologically predominant, limited structural information is known about those species. Nanoelectrospray ion mobility mass spectrometry (nESI-IM-MS) provides another possibility to study apo- and partially metalated-MT. We presented here for the first report to apply collision-induced unfolding (CIU) to study the gas-phase stabilities of MTs after partially metalated by Cd^2+, Zn^2+ and Ag^+ . Cdv4-MT, Znv4-MT, Agv4-MT and Agv6-MT differ dramatically in their gas-phase stabilities. A static mixing tee with variable lengths of capillary was chosen to probe abundance change of partially metalated species of MT binding with Cd^2+ and Zn^2+ during short reaction time. The mixing tee system also allows CIU study of intermediates under preequilibrium conditions. The sequential addition of each Cd^2+ and Zn^2+ ion to MT results in the incremental stabilization of unique unfolding intermediates. We further focused on Ag^+ metalated MT and combined multiple MS strategies, including mixed metalation, CIU, chemical labeling, top-down and bottom-up MS, and 2D MS-CID-IM-MS, to unambiguously identify the binding sites of Agv4-MT. Both Cd^2+ and Ag^+ bind to MT cooperatively, however, Cdv4-MT prefers binding to α domain while Agv4-MT binds to β domain.
Furthermore, some attempts including kinetic study with mixing tee during Cd^2+ metalation, Cu^+ binding to MT and studies related to possible solution phase structures of different charge states of MT are summarized. We hope those attempts can inspire future study. | en |
