Applying Collision-Induced Unfolding to Study Gas Phase Stability of Partially Metalated Metallothionein-2A
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.
Citation
Dong, Shiyu (2020). Applying Collision-Induced Unfolding to Study Gas Phase Stability of Partially Metalated Metallothionein-2A. Doctoral dissertation, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /191618.