Earthworm Lumbricus rubellus MT-2: Metal Binding and Protein Folding of a True Cadmium-MT.
Earthworm Lumbricus rubellus MT-2: Metal Binding and Protein Folding of a True Cadmium-MT.
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DOI：
10.3390/ijms17010065
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发表时间：
2016-01-05
影响因子：
5.6
通讯作者：
Blindauer CA
中科院分区：
文献类型：
--
作者：
Kowald GR;Stürzenbaum SR;Blindauer CA
蚯蚓表达，大多数动物，金属硫蛋白（MT）-小，半胱氨酸丰富的蛋白质结合d10金属离子（锌（II），镉（II），或铜（I））在集群。已知三种MT同系物存在于红蚯蚓中，其中一种是wMT-2，它是由蠕虫暴露于镉强烈诱导的。本研究涉及的组合物，金属结合亲和力和金属依赖的蛋白质折叠的wMT-2重组表达和纯化的存在下，镉（II）和锌（II）。重要的是，虽然从表达wMT-2的E.大肠杆菌培养物补充镉（II），在锌（II）的存在下表达产生的混合物。测定的镉（II）或锌（II）的wMT-2的平均亲和力都在MT的正常范围内;因此，差异行为不能解释的基础上的整体亲和力。因此，通过1H NMR光谱比较了Cd-和Zn-wMT-2的蛋白质折叠性质。这种比较表明，在镉的存在下比在锌的存在下更好地定义了蛋白质折叠。折叠和动力学的这些差异可能是镉和锌结合蛋白在体外的差异行为的根源，并可能最终也有助于区分锌和镉在体内的细胞中。
Earthworms express, as most animals, metallothioneins (MTs)—small, cysteine-rich proteins that bind d10 metal ions (Zn(II), Cd(II), or Cu(I)) in clusters. Three MT homologues are known for Lumbricus rubellus, the common red earthworm, one of which, wMT-2, is strongly induced by exposure of worms to cadmium. This study concerns composition, metal binding affinity and metal-dependent protein folding of wMT-2 expressed recombinantly and purified in the presence of Cd(II) and Zn(II). Crucially, whilst a single Cd7wMT-2 species was isolated from wMT-2-expressing E. coli cultures supplemented with Cd(II), expressions in the presence of Zn(II) yielded mixtures. The average affinities of wMT-2 determined for either Cd(II) or Zn(II) are both within normal ranges for MTs; hence, differential behaviour cannot be explained on the basis of overall affinity. Therefore, the protein folding properties of Cd- and Zn-wMT-2 were compared by 1H NMR spectroscopy. This comparison revealed that the protein fold is better defined in the presence of cadmium than in the presence of zinc. These differences in folding and dynamics may be at the root of the differential behaviour of the cadmium- and zinc-bound protein in vitro, and may ultimately also help in distinguishing zinc and cadmium in the earthworm in vivo.