Non-toxic bismuth salts are used in anti-ulcer medications and to protect against nephrotoxicity from anticancer drugs. Bismuth salts also induce metallothionein (MT), a metal-binding protein that lacks a formal secondary structure. We report the impact on the metallation properties of Bi(III) to the 9-cysteine β fragment of MT as a function of cysteine accessibility using electrospray ionization mass spectrometry. At pH 7.4, Bi2βMT formed cooperatively. Cysteine modification shows that each Bi(III) was terminally bound to three cysteinyl thiolates. Non-cooperative Bi(III) binding was observed at pH 2.3, where cysteine accessibility is increased. However, competition from H4EDTA inhibited Bi(III) binding. When GdmCl, a well-known denaturing agent, was used to increase cysteine accessibility of the apoβMT at pH 7.4, a greater fraction of Bi3βMT formed using all nine cysteines. The change in binding profile and equilibrium of Bi2βMT was determined as a function of acidification, which changed as a result of competition with H4EDTA. There was no Bi(III) transfer between Bi2βMT, Cd3βMT, and Zn3βMT. This lack of metal exchange and the resistance towards binding the third Bi(III) suggest a rigidity in the Bi2βMT binding sites that inhibits Bi(III) mobility. These experiments emphasize the conformational control of metallation that results in substantially different metallated products: at pH 7.4 (many cysteines buried) Bi2βMT, whereas at pH 7.4 (all cysteines accessible) enhanced formation of Bi3βMT. These data suggest that the addition of the first two Bi(III) crosslinks the protein, blocking access to the remaining three cysteines for the third Bi(III), as a result of tangle formation.

中文翻译：

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apo-βMT1a 的结构受限的 Bi(III) 金属化：金属诱导的缠结。

无毒的铋盐用于抗溃疡药物和防止抗癌药物的肾毒性。铋盐还诱导金属硫蛋白 (MT)，这是一种缺乏正式二级结构的金属结合蛋白。我们使用电喷雾电离质谱报告了作为半胱氨酸可及性的函数对 Bi(III) 对 MT 的 9-半胱氨酸 β 片段的金属化特性的影响。在 pH 7.4 时，Bi2βMT 协同形成。半胱氨酸修饰显示每个 Bi(III) 末端与三个半胱氨酸硫醇盐结合。在 pH 2.3 下观察到非合作性 Bi(III) 结合，其中半胱氨酸可及性增加。然而，来自 H4EDTA 的竞争抑制了 Bi(III) 的结合。当使用众所周知的变性剂 GdmCl 在 pH 7.4 时增加 apoβMT 的半胱氨酸可及性时，使用所有九个半胱氨酸形成更大比例的 Bi3βMT。Bi2βMT 结合谱和平衡的变化被确定为酸化的函数，由于与 H4EDTA 竞争而发生变化。Bi2βMT、Cd3βMT 和 Zn3βMT 之间没有 Bi(III) 转移。这种缺乏金属交换和对结合第三个 Bi(III) 的抵抗表明 Bi2βMT 结合位点的刚性抑制了 Bi(III) 的迁移。这些实验强调了金属化的构象控制，这导致了显着不同的金属化产物：在 pH 7.4（许多半胱氨酸被掩埋）时 Bi2βMT，而在 pH 7.4（所有半胱氨酸均可用）增强了 Bi3βMT 的形成。这些数据表明，前两个 Bi(III) 的添加使蛋白质交联，阻止了第三个 Bi(III) 进入剩余的三个半胱氨酸，