High-Throughput PIXE as an Essential Quantitative Assay for Accurate Metalloprotein Structural Analysis: Development and Application
High-Throughput PIXE as an Essential Quantitative Assay for Accurate Metalloprotein Structural Analysis: Development and Application
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DOI：
10.1021/jacs.9b09186
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发表时间：
2020-01-08
影响因子：
15
通讯作者：
Garman, Elspeth F.
中科院分区：
文献类型：
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作者：
Grime, Geoffrey W.;Zeldin, Oliver B.;Garman, Elspeth F.
金属蛋白质占蛋白质的三分之一以上，大约一半的酶需要金属才能发挥作用。准确识别这些金属原子及其环境是理解生物机制的先决条件。使用离子束分析，通过粒子诱导X射线发射（PIXE），我们已经定量确定了金属原子在30个以前的结构特征的蛋白质，使用最小的样品体积和高通量的方法。这些金属中有一半以上在沉积结构模型中被错误识别。一些PIXE检测到的金属没有看到的模型中被解释为文物混杂结晶试剂。对于其他人来说，使用正确的金属改善了结构模型。对于多核位点，异常衍射信号使正确金属的定位能够揭示先前模糊的生物信息。PIXE对化学环境不敏感，但与与结构模型一起沉积的实验衍射数据相结合，它可以验证和潜在的金属蛋白模型修复，改善结构，更重要的是，机械知识。
Metalloproteins comprise over one-third of proteins, with approximately half of all enzymes requiring metal to function. Accurate identification of these metal atoms and their environment is a prerequisite to understanding biological mechanism. Using ion beam analysis through particle induced X-ray emission (PIXE), we have quantitatively identified the metal atoms in 30 previously structurally characterized proteins using minimal sample volume and a high-throughput approach. Over half of these metals had been misidentified in the deposited structural models. Some of the PIXE detected metals not seen in the models were explainable as artifacts from promiscuous crystallization reagents. For others, using the correct metal improved the structural models. For multinuclear sites, anomalous diffraction signals enabled the positioning of the correct metals to reveal previously obscured biological information. PIXE is insensitive to the chemical environment, but coupled with experimental diffraction data deposited alongside the structural model it enables validation and potential remediation of metalloprotein models, improving structural and, more importantly, mechanistic knowledge.