Site-specific resolution of anionic residues in proteins using solid-state NMR spectroscopy
Site-specific resolution of anionic residues in proteins using solid-state NMR spectroscopy
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DOI：
10.1007/s10858-020-00323-z
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发表时间：
2020-06
影响因子：
2.7
通讯作者：
Jianping Li;Ampon Sae Her;N. Traaseth
中科院分区：
文献类型：
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作者：
Jianping Li;Ampon Sae Her;N. Traaseth
由于化学位移对质子化状态很敏感，因此核磁共振光谱通常用于推断特定位置的酸解离常数（pKa）。探测最接近酸/碱化学中官能团的原子的方法对于确定质子化状态是最敏感的。在这项工作中，我们描述了一种魔角旋转（MAS）固态核磁共振方法来测量阴离子残基天冬氨酸和谷氨酸侧链上的化学位移。该方法结合了间接维双量子光谱和REDOR脱相，提供了这些氨基酸残基的敏感和分辨率视图，这些氨基酸残基通常参与酶催化和膜蛋白运输。为了证明该方法的适用性，我们使用微晶可溶性蛋白（泛素）和嵌入脂质双层的膜蛋白（EmrE）进行了测量。总的来说，双量子维度的分辨率和对从REDOR过滤器中识别天冬氨酸和谷氨酸残基的信心使该方法最方便地表征质子化状态和使用MAS固态核磁共振获得pkk值。
NMR spectroscopy is commonly used to infer site-specific acid dissociation constants (pKa) since the chemical shift is sensitive to the protonation state. Methods that probe atoms nearest to the functional groups involved in acid/base chemistry are the most sensitive for determining the protonation state. In this work, we describe a magic-angle-spinning (MAS) solid-state NMR approach to measure chemical shifts on the side chain of the anionic residues aspartate and glutamate. This method involves a combination of double quantum spectroscopy in the indirect dimension and REDOR dephasing to provide a sensitive and resolved view of these amino acid residues that are commonly involved in enzyme catalysis and membrane protein transport. To demonstrate the applicability of the approach, we carried out measurements using a microcrystalline soluble protein (ubiquitin) and a membrane protein embedded in lipid bilayers (EmrE). Overall, the resolution available from the double quantum dimension and confidence in identification of aspartate and glutamate residues from the REDOR filter make this method the most convenient for characterizing protonation states and deriving pKavalues using MAS solid-state NMR.