Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK.
Phys Chem Chem Phys. 2010 Nov 7;12(41):13408-18. doi: 10.1039/c0cp00680g. Epub 2010 Sep 27.
The direct observation of binding and release of spectroscopically silent metal ions such as Zn(2+) and Ca(2+) by proteins has been challenging before the advent of native electrospray ionisation mass spectrometry. This report highlights the powerful capability of ESI-MS to provide insight into metalloprotein speciation that is independent of any spectroscopic property. Using the zinc-binding plant metallothionein E(C) from wheat as a study case, we show that ESI-MS is unique amongst other techniques in capturing intermediary metallospecies that evolve during the course of metal transfer to the chelator EDTA, as a model reaction to mimic the biological function of the protein as a zinc donor. Zinc release from the two-domain protein E(C) appears to be extremely rapid and non-cooperative, and progresses with loss of one zinc ion from the fully loaded Zn(6) species, and a transient build-up of Zn(5) and Zn(4) species, which further react to give species with 0-3 zinc ions bound. (1)H NMR data has provided further insights into the different behaviour of the two domains upon metal depletion.
在原生电喷雾电离质谱出现之前，直接观察光谱静默金属离子（如 Zn(2+)和 Ca(2+)）与蛋白质的结合和释放一直具有挑战性。本报告强调了 ESI-MS 提供独立于任何光谱特性的金属蛋白形态分析的强大能力。本文以小麦锌结合植物金属硫蛋白 E(C)作为研究案例，表明 ESI-MS 在捕获金属转移到螯合剂 EDTA 过程中演变的中间金属物种方面具有独特的能力，这是模拟蛋白质作为锌供体的生物学功能的模型反应。从两域蛋白 E(C)中释放锌似乎非常迅速且非协同，并且随着完全加载的 Zn(6)物种中一个锌离子的丢失以及 Zn(5)和 Zn(4)物种的短暂积累而进行，进一步反应生成具有 0-3 个锌离子结合的物种。（1）H NMR 数据进一步深入了解了金属耗尽时两个结构域的不同行为。
