Instituto de Tecnologia Química e Biológica António Xavier (ITQB-NOVA), Universidade Nova de Lisboa, Oeiras, Portugal.
Magnetic Resonance Center and Department of Chemistry, University of Florence, Sesto Fiorentino, Italy.
FEBS J. 2021 May;288(9):3010-3023. doi: 10.1111/febs.15615. Epub 2020 Nov 23.
Metalloproteins play key roles across biology, and knowledge of their structure is essential to understand their physiological role. For those metalloproteins containing paramagnetic states, the enhanced relaxation caused by the unpaired electrons often makes signal detection unfeasible near the metal center, precluding adequate structural characterization right where it is more biochemically relevant. Here, we report a protein structure determination by NMR where two different sets of restraints, one containing Nuclear Overhauser Enhancements (NOEs) and another containing Paramagnetic Relaxation Enhancements (PREs), are used separately and eventually together. The protein PioC from Rhodopseudomonas palustris TIE-1 is a High Potential Iron-Sulfur Protein (HiPIP) where the [4Fe-4S] cluster is paramagnetic in both oxidation states at room temperature providing the source of PREs used as alternative distance restraints. Comparison of the family of structures obtained using NOEs only, PREs only, and the combination of both reveals that the pairwise root-mean-square deviation (RMSD) between them is similar and comparable with the precision within each family. This demonstrates that, under favorable conditions in terms of protein size and paramagnetic effects, PREs can efficiently complement and eventually replace NOEs for the structural characterization of small paramagnetic metalloproteins and de novo-designed metalloproteins by NMR. DATABASES: The 20 conformers with the lowest target function constituting the final family obtained using the full set of NMR restraints were deposited to the Protein Data Bank (PDB ID: 6XYV). The 20 conformers with the lowest target function obtained using NOEs only (PDB ID: 7A58) and PREs only (PDB ID: 7A4L) were also deposited to the Protein Data Bank. The chemical shift assignments were deposited to the BMRB (code 34487).
金属蛋白在生物学中起着关键作用，了解它们的结构对于理解它们的生理功能至关重要。对于那些含有顺磁态的金属蛋白，未配对电子引起的增强弛豫作用常常使得在金属中心附近无法进行信号检测，从而无法在更具生物化学相关性的地方进行充分的结构特征描述。在这里，我们报告了一种通过 NMR 进行的蛋白质结构测定方法，其中使用了两组不同的约束条件，一组包含核 Overhauser 增强（NOE），另一组包含顺磁弛豫增强（PRE），分别使用并最终一起使用。来自沼泽红假单胞菌 TIE-1 的 PioC 蛋白是一种高潜在铁硫蛋白（HiPIP），其中[4Fe-4S]簇在室温下的两种氧化态都是顺磁的，为使用 PRE 作为替代距离约束的来源提供了条件。使用仅 NOE、仅 PRE 和两者组合获得的结构族的比较表明，它们之间的两两均方根偏差（RMSD）相似，并且与每个家族内的精度相当。这表明，在蛋白质大小和顺磁效应方面的有利条件下，PRE 可以有效地补充并最终取代 NOE，用于通过 NMR 对小顺磁金属蛋白和从头设计的金属蛋白进行结构特征描述。数据库：使用完整的 NMR 约束条件获得的最终结构族中具有最低目标函数的 20 个构象已被存入蛋白质数据库（PDB ID：6XYV）。仅使用 NOE（PDB ID：7A58）和仅使用 PRE（PDB ID：7A4L）获得的具有最低目标函数的 20 个构象也被存入蛋白质数据库。化学位移分配已存入 BMRB（代码 34487）。
