Institute of Chemistry, Eötvös Loránd University, Pázmány Péter sétány 1/a, Budapest 1117, Hungary.
Institut für Organische Chemie and Institut für Biologische Grenzflächen 4-Magnetische Resonanz, Karlsruher Institut für Technologie (KIT), Fritz-Haber-Weg 6, Karlsruhe 76133, Germany.
Anal Chem. 2020 Sep 15;92(18):12423-12428. doi: 10.1021/acs.analchem.0c02182. Epub 2020 Aug 31.
Intrinsically disordered proteins (IDPs) constitute an important class of biomolecules with high flexibility. Atomic-resolution studies for these molecules are essentially limited to NMR spectroscopy, which should be performed under physiological pH and temperature to populate relevant conformational ensembles. In this context, however, fundamental problems arise with established triple resonance NMR experiments: high solvent accessibility of IDPs promotes water exchange, which disfavors classical amide H-detection, while C-detection suffers from significantly reduced sensitivity. A favorable alternative, the conventional detection of nonexchangeable Hα, so far resulted in broad signals with insufficient resolution and sensitivity. To overcome this, we introduce here a selective Hα,Cα-correlating pure shift detection scheme, the selective Hα,Cα-HSQC (SHACA-HSQC), using extensive hetero- and homonuclear decoupling applicable to aqueous samples (≥90% HO) and tested on small molecules and proteins. SHACA-HSQC spectra acquired on IDPs provide uncompromised resolution and sensitivity (up to fivefold increased S/N compared to the standard H,C-HSQC), as shown for resonance distinction and unambiguous assignment on the disordered transactivation domain of the tumor suppressor p53, α-synuclein, and folded ubiquitin. The detection scheme can be implemented in any Hα-detected triple resonance experiment and may also form the basis for the detection of isotope-labeled markers in biological studies or compound libraries.
无规卷曲蛋白质（IDPs）构成了具有高灵活性的重要生物分子类群。这些分子的原子分辨率研究本质上仅限于 NMR 光谱学，该方法应在生理 pH 和温度下进行，以形成相关的构象集合。然而，在这种情况下，建立的三重共振 NMR 实验会出现一些基本问题：IDPs 的高溶剂可及性促进了水交换，这不利于经典酰胺 H 检测，而 C 检测的灵敏度则显著降低。一种有利的替代方法是传统的不可交换 Hα检测，迄今为止，它产生了具有足够分辨率和灵敏度的宽信号。为了克服这个问题，我们在这里引入了一种选择性的 Hα，Cα 相关的纯移检测方案，即选择性的 Hα，Cα-HSQC（SHACA-HSQC），使用广泛的异核和同核去耦，适用于水相样品（≥90%HO），并在小分子和蛋白质上进行了测试。在 IDPs 上获得的 SHACA-HSQC 谱提供了不妥协的分辨率和灵敏度（与标准的 H，C-HSQC 相比，S/N 增加了高达五倍），如在肿瘤抑制因子 p53 的无序转录激活结构域、α-突触核蛋白和折叠泛素上的共振区分和明确分配中所示。该检测方案可在任何 Hα 检测的三重共振实验中实施，也可作为生物研究或化合物库中同位素标记标记物检测的基础。
