Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle-Strasse 10, 13125, Berlin, Germany.
Institut für Chemie und Biochemie, Freie Universität Berlin, Takustrasse 3, 14195, Berlin, Germany.
J Biomol NMR. 2020 May;74(4-5):247-256. doi: 10.1007/s10858-020-00307-z. Epub 2020 Mar 17.
Hydrogen bonds are essential for protein structure and function, making experimental access to long-range interactions between amide protons and heteroatoms invaluable. Here we show that measuring distance restraints involving backbone hydrogen atoms and carbonyl- or α-carbons enables the identification of secondary structure elements based on hydrogen bonds, provides long-range contacts and validates spectral assignments. To this end, we apply specifically tailored, proton-detected 3D (H)NCOH and (H)NCAH experiments under fast magic angle spinning (MAS) conditions to microcrystalline samples of SH3 and GB1. We observe through-space, semi-quantitative correlations between protein backbone carbon atoms and multiple amide protons, enabling us to determine hydrogen bonding patterns and thus to identify β-sheet topologies and α-helices in proteins. Our approach shows the value of fast MAS and suggests new routes in probing both secondary structure and the role of functionally-relevant protons in all targets of solid-state MAS NMR.
氢键对于蛋白质结构和功能至关重要，因此实验获取酰胺质子和杂原子之间的长程相互作用是非常宝贵的。在这里，我们展示了测量涉及骨架氢原子和羰基或α-碳原子的距离约束可以基于氢键识别二级结构元件，提供长程接触并验证光谱分配。为此，我们在快速魔角旋转（MAS）条件下应用专门定制的质子探测 3D（H）NCOH 和（H）NCAH 实验，对 SH3 和 GB1 的微晶样品进行实验。我们观察到蛋白质骨架碳原子和多个酰胺质子之间的空间相关，这使我们能够确定氢键模式，从而确定蛋白质中的β-折叠拓扑结构和α-螺旋。我们的方法展示了快速 MAS 的价值，并为探测所有固态 MAS NMR 靶标中的二级结构和功能相关质子的作用提供了新的途径。
