Leibniz Institute for Molecular Pharmacology, Robert Roessle Str. 10, 13125 Berlin (Germany).
Angew Chem Int Ed Engl. 2014 Feb 24;53(9):2438-42. doi: 10.1002/anie.201308927. Epub 2014 Jan 29.
(1)H-detected magic-angle spinning NMR experiments facilitate structural biology of solid proteins, which requires using deuterated proteins. However, often amide protons cannot be back-exchanged sufficiently, because of a possible lack of solvent exposure. For such systems, using (2)H excitation instead of (1)H excitation can be beneficial because of the larger abundance and shorter longitudinal relaxation time, T1, of deuterium. A new structure determination approach, "quadruple-resonance NMR spectroscopy", is presented which relies on an efficient (2)H-excitation and (2)H-(13)C cross-polarization (CP) step, combined with (1)H detection. We show that by using (2)H-excited experiments better sensitivity is possible on an SH3 sample recrystallized from 30 % H2O. For a membrane protein, the ABC transporter ArtMP in native lipid bilayers, different sets of signals can be observed from different initial polarization pathways, which can be evaluated further to extract structural properties.
(1)H 检测魔角旋转 NMR 实验促进了固态蛋白质的结构生物学研究，这需要使用氘代蛋白质。然而，由于可能缺乏溶剂暴露，酰胺质子往往不能充分回交。对于此类系统，由于氘的丰度较大且纵向弛豫时间 T1 较短，使用 (2)H 激发而不是 (1)H 激发可能会更有利。本文提出了一种新的结构确定方法“四重共振 NMR 光谱学”，它依赖于有效的 (2)H 激发和 (2)H-(13)C 交叉极化 (CP) 步骤，以及 (1)H 检测。我们表明，通过使用 (2)H 激发实验，在从 30% H2O 重结晶的 SH3 样品上可以获得更好的灵敏度。对于一种膜蛋白，即天然脂质双层中的 ABC 转运蛋白 ArtMP，可以从不同的初始极化途径观察到不同的信号集，这些信号集可以进一步评估以提取结构特性。
