Correlation Spectra in MAS Solid-State NMR using Deuterated Proteins with Selective 1 H , 2 H Isotopic Labeling of Methyl Groups
Correlation Spectra in MAS Solid-State NMR using Deuterated Proteins with Selective 1 H , 2 H Isotopic Labeling of Methyl Groups
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使用具有甲基选择性 1 H 、 2 H 同位素标记的氘化蛋白质的 MAS 固态 NMR 中的相关光谱
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发表时间：
2006
期刊：
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通讯作者：
B. Reif
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作者：
Vipin Agarwal;A. Diehl;N. Skrynnikov;B. Reif
魔角自旋（MAS）固态核磁共振（ssNMR）在过去几年中发展迅速。这一发展导致了淀粉样蛋白生成肽1 -3和膜蛋白的第一个结构模型。4测定这些结构的一个主要障碍是ssNMR实验的低信噪比。到目前为止，所有标准ssNMR实验都依赖于异核检测（13 C/15 N）。原则上，质子由于其大的旋磁比（γH）而更适合。然而，由于质子耦合网络的均匀性，即使在存在线窄化技术（如魔角旋转（MAS）和homopolar偶极去耦）的情况下，大的γH也意味着大的1H，1H偶极耦合。5蛋白质的氘化和质子对可交换的氘核的回取代允许降低有效的1H，1H偶极耦合并促进ssNMR脉冲序列的发展。6-8我们最近可以证明，在结晶缓冲液中使用90%D2O进一步降低质子自旋密度，导致1H线宽减少4倍（与从100%H2O中重结晶的样品相比）。在24 kHz的MAS频率下，所获得的HN线宽为20 Hz的量级。然而，这种方法由于质子数量减少而导致灵敏度损失。在这项试点研究中提出的方法避免了这个陷阱，如凯和同事最初提出的甲基基团的标记。10
Magic angle spinning (MAS) solid-state nuclear magnetic resonance (ssNMR) has developed rapidly over the past few years. This development led to the first structural models of amyloidogenic peptides1-3 and membrane proteins. 4 A major obstacle in the determination of these structures is the low signal-to-noise ratio associated with ssNMR experiments. So far, all standard ssNMR experiments have relied on heteronucleus detection ( 13C/15N). Protons would, in principle, be better suited due to their large gyromagnetic ratio ( γH). However, a largeγH implicates large1H,1H dipolar couplings even in the presence of line narrowing techniques, such as magic angle spinning (MAS) and homonuclear dipolar decoupling, because of the homogeneous nature of the proton coupling network. 5 Deuteration of a protein and back-substitution of exchangeable deuterons by protons allows one to decrease the effective 1H,1H dipolar couplings and facilitates ssNMR pulse sequence development. 6-8 We could show recently 9 that a further decrease in the proton spin density employing 90% D2O in the crystallization buffer sresulted in a 4-fold reduction in the 1H line width (compared to a sample recrystallized from 100% H 2O). The obtained HN line width was on the order of 20 Hz at a MAS frequency of 24 kHz. However, this approach induces a loss in sensitivity due to the decreased number of protons. The approach which is presented in this pilot study avoids this pitfall by labeling of methyl groups as originally proposed by Kay and co-workers. 10