Dynamics of iron release from transferrin N-lobe studied by electrospray ionization mass spectrometry
Dynamics of iron release from transferrin N-lobe studied by electrospray ionization mass spectrometry
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DOI：
10.1021/ac0015164
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发表时间：
2001-06-01
影响因子：
7.4
通讯作者：
Kaltashov, IA
中科院分区：
文献类型：
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作者：
Gumerov, DR;Kaltashov, IA
转铁蛋白构成一类金属蛋白，参与多种物种的循环铁转运。由于这些蛋白质在各种生物和医疗保健相关领域极其重要，它们的金属离子结合特性在过去十年中一直是广泛研究工作的焦点。这些蛋白质的大尺寸以及高自旋金属离子（例如 Fe3+）的存在限制了 NMR 的使用。在这项工作中，我们报告了使用电喷雾电离质谱 (ESI MS) 在模拟内体环境的条件下体外研究转铁蛋白系统的动力学。 ESI MS 可以为转铁蛋白结合/释放金属离子的机制提供有价值的见解，并且是其他光谱技术的补充。根据蛋白质离子电荷态分布的出现来评估复合物的构象稳定性，而根据蛋白质离子的质量确定蛋白质-配体复合物的组成。在不存在铁螯合剂的情况下，随着溶液 pH 值逐渐降低，观察到三元复合物（蛋白质-金属离子-协同阴离子）的逐步解离。虽然协同阴离子从复合物中的释放是在典型的内体 pH 水平（即 5.5）下开始的，但金属离子仍然很大程度上与蛋白质结合，直到 pH 值降低到类似于 4.5 的水平。在这些条件下，很大一部分蛋白质形成未折叠构象。与这种行为形成鲜明对比的是，向蛋白质溶液中添加铁螯合剂（柠檬酸盐）会导致铁在典型的内体 pH 水平下轻松释放，而不会检测到任何蛋白质的未折叠。质谱数据进一步证明了以下观点的可信度：全蛋白采样了载脂蛋白形式特有的构象（例如“开放构象”），铁最有可能从该构象中发生解离。本研究的结果表明，ESI MS 可用于模拟在模拟生理环境的条件下从转铁蛋白释放的金属离子。
Transferrins constitute a class of metalloproteins that are involved in circulatory iron transport in a variety of species. The metal ion-binding properties of these proteins have been die focus of extensive research efforts in the past decade due to their extreme importance in a variety of biological and healthcare-related fields. The large size of these proteins, as well as the presence of high-spin metal ions (e.g,, Fe3+), limits the use of NMR, In this work, we report on the use of electrospray ionization mass spectrometry (ESI MS) to study dynamics of the transferrin system in vitro under conditions that are designed to mimic the endosomal environment. ESI MS is shown to provide valuable insights into the mechanistic aspects of metal ion-binding/release by transferrins and is complementary to other spectroscopic techniques. Conformational stability of the complex is evaluated based on the appearance of the charge-state distribution of protein ions, while the composition of the protein-ligand complex is determined based on the mass of the protein ions. In the absence of iron chelators, a stepwise dissociation of the ternary complex (protein-metal ion-synergistic anion) is observed as the solution pH is gradually decreased. Although the release of synergistic anion from the complex is initiated at typical endosomal pH levels (i.e., 5.5), metal ion remains largely bound to the protein until the pH is lowered to a level of similar to4.5. Under these conditions, a significant fraction of the protein populates unfolded conformations, In stark contrast to this behavior, addition of an iron chelating agent (citrate) to the protein solution results in facile iron release at typical endosomal pH levels without any detectable unfolding of the protein. The mass spectral data lends further credibility to the notion that the holoprotein samples conformations that are specific to the apo form (e.g, "open conformation"), from which iron dissociation most likely occurs. The results of the present study demonstrate that ESI MS can be used to model metal ion release from transferrin under conditions that are designed to mimic the physiological environment.