Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.
Anal Chem. 2020 Jul 7;92(13):8923-8932. doi: 10.1021/acs.analchem.0c00829. Epub 2020 Jun 24.
Metallothioneins (MTs) constitute a family of cysteine-rich proteins that play key biological roles for a wide range of metal ions, but unlike many other metalloproteins, the structures of apo- and partially metalated MTs are not well understood. Here, we combine nano-electrospray ionization-mass spectrometry (ESI-MS) and nano-ESI-ion mobility (IM)-MS with collision-induced unfolding (CIU), chemical labeling using -ethylmaleimide (NEM), and both bottom-up and top-down proteomics in an effort to better understand the metal binding sites of the partially metalated forms of human MT-2A, viz., Ag-MT. The results for Ag-MT are then compared to similar results obtained for Cd-MT. The results show that Ag-MT is a cooperative product, and data from top-down and bottom-up proteomics mass spectrometry analysis combined with NEM labeling revealed that all four Ag ions of Ag-MT are bound to the β-domain. The binding sites are identified as Cys13, Cys15, Cys19, Cys21, Cys24, and Cys26. While both Ag and Cd react with MT to yield cooperative products, i.e., Ag-MT and Cd-MT, these products are very different; Ag ions of Ag-MT are located in the β-domain, whereas Cd ions of Cd-MT are located in the α-domain. Ag-MT has been reported to be fully metalated in the β-domain, but our data suggest the two additional Ag ions are more weakly bound than are the other four. Higher order Ag-MT complexes ( = 7-17) are formed in solutions that contain excess Ag ions, and these are assumed to be bound to the α-domain or shared between the two domains. Interestingly, the excess Ag ions are displaced upon addition of NEM to this solution to yield predominantly AgNEM-MT. Results from CIU suggest that Ag-MT complexes are structurally more ordered and that the energy required to unfold these complexes increases as the number of coordinated Ag increases.
金属硫蛋白（MTs）构成一组富含半胱氨酸的蛋白质，它们在广泛的金属离子中发挥关键的生物学作用，但与许多其他金属蛋白不同，去辅基和部分金属化的 MTs 的结构尚未得到很好的理解。在这里，我们将纳喷雾电离 - 质谱（ESI-MS）和纳电喷雾离子迁移率（IM）-MS 与碰撞诱导展开（CIU）、使用 - 乙基马来酰亚胺（NEM）的化学标记以及自上而下和自下而上的蛋白质组学相结合，以更好地理解部分金属化形式的人类 MT-2A，即 Ag-MT 的金属结合位点。然后将 Ag-MT 的结果与 Cd-MT 获得的类似结果进行比较。结果表明，Ag-MT 是一种协同产物，来自自上而下和自下而上的蛋白质组学质谱分析以及 NEM 标记的数据表明，Ag-MT 的所有四个 Ag 离子都与β-结构域结合。结合位点被鉴定为 Cys13、Cys15、Cys19、Cys21、Cys24 和 Cys26。虽然 Ag 和 Cd 都与 MT 反应生成协同产物，即 Ag-MT 和 Cd-MT，但这些产物却大不相同；Ag-MT 的 Ag 离子位于β-结构域，而 Cd-MT 的 Cd 离子位于α-结构域。据报道，Ag-MT 在β-结构域中完全金属化，但我们的数据表明，另外两个 Ag 离子的结合比其他四个 Ag 离子弱。在含有过量 Ag 离子的溶液中形成更高阶的 Ag-MT 复合物（=7-17），并假定这些复合物与α-结构域或两个结构域之间结合。有趣的是，在向该溶液中加入 NEM 时，会取代多余的 Ag 离子，从而主要产生 AgNEM-MT。CIU 的结果表明，Ag-MT 复合物在结构上更有序，并且随着配位 Ag 数量的增加，展开这些复合物所需的能量也会增加。
