NMR spectroscopy is commonly used to infer site-specific acid dissociation constants (pK_a) 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 pK_a values using MAS solid-state NMR.

NMR光谱通常用于推断位点特定的酸解离常数（p K _a），因为化学位移对质子化状态敏感。探测最接近酸/碱化学中涉及的官能团的原子的方法对于确定质子化状态最敏感。在这项工作中，我们描述了一种魔角旋转（MAS）固态NMR方法，用于测量阴离子残基天冬氨酸和谷氨酸的侧链上的化学位移。此方法涉及间接维度上的双量子光谱和REDOR移相的组合，以提供对这些通常在酶催化和膜蛋白运输中涉及的氨基酸残基的灵敏且可分辨的视图。为了证明该方法的适用性，我们使用微晶可溶性蛋白（泛素）和包埋在脂质双层中的膜蛋白（EmrE）进行了测量。总体，使用MAS固态NMR的K _a值。