Department of Biology, Chemistry, Pharmacy, Freie Universität Berlin, 14195 Berlin, Germany.
Analyst. 2016 Oct 7;141(19):5502-10. doi: 10.1039/c6an01377e. Epub 2016 Aug 5.
In this study the gas-phase structure of ubiquitin and its lysine-to-arginine mutants was investigated using ion mobility-mass spectrometry (IM-MS) and electron transfer dissociation-mass spectrometry (ETD-MS). Crown ether molecules were attached to positive charge sites of the proteins and the resulting non-covalent complexes were analysed. Collision induced dissociation (CID) experiments revealed relative energy differences between the wild type and the mutant crown-ether complexes. ETD-MS experiments were performed to identify the crown ether binding sites. Although not all of the binding sites could be revealed, the data confirm that the first crown ether is able to bind to the N-terminus. IM-MS experiments show a more compact structure for specific charge states of wild type ubiquitin when crown ethers are attached. However, data on ubiquitin mutants reveal that only specific lysine residues contribute to the effect of charge microsolvation. A compaction is only observed for one of the investigated mutants, in which the lysine has no proximate interaction partner. On the other hand when the lysine residues are involved in salt bridges, attachment of crown ethers has little effect on the structure.
在这项研究中，使用离子淌度-质谱（IM-MS）和电子转移解离-质谱（ETD-MS）研究了泛素及其赖氨酸到精氨酸突变体的气相结构。冠醚分子被连接到蛋白质的正电荷位点上，并分析了由此产生的非共价复合物。碰撞诱导解离（CID）实验揭示了野生型和突变体冠醚复合物之间的相对能量差异。进行 ETD-MS 实验以确定冠醚结合位点。尽管不能揭示所有的结合位点，但数据证实第一个冠醚能够结合到 N 端。IM-MS 实验表明，当连接冠醚时，野生型泛素的特定电荷状态具有更紧凑的结构。然而，关于泛素突变体的数据表明，只有特定的赖氨酸残基有助于电荷微溶剂化的影响。只有在一个被研究的突变体中观察到紧凑，其中赖氨酸没有接近的相互作用伙伴。另一方面，当赖氨酸残基参与盐桥时，冠醚的附着对结构几乎没有影响。
