Methyl-TROSY based NMR experiments have over the last two decades become one of the most important means to characterise dynamics and functional mechanisms of large proteins and macromolecular machines in solution. The chemical shift assignment of methyl groups in large proteins is, however, still not trivial and it is typically performed using backbone-dependent experiments in a 'divide and conquer' approach, mutations, structure-based assignments or a combination of these. Structure-based assignment of methyl groups is an emerging strategy, which reduces the time and cost required as well as providing a method that is independent of a backbone assignment. One crucial step in available structure-based assignment protocols is linking the two prochiral methyl groups of leucine and valine residues. This has previously been achieved by recording NOESY spectra with short mixing times or by comparing NOESY spectra. Herein, we present a method based on through-bond scalar coupling transfers, a 3D-HMBC-HMQC experiment, to link the intra-residue methyl groups of leucine and valine. It is shown that the HMBC-HMQC method has several advantages over solely using NOESY spectra since a unique intra-residue cross-peak is observed. Moreover, overlap in the methyl-TROSY HMQC spectrum can easily be identified with the HMBC-HMQC experiment, thereby removing possible ambiguities in the assignment.

中文翻译：

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来自3D-HMBC-HMQC实验的大蛋白中缬氨酸和亮氨酸残基的残基甲基-甲基相关性。

在过去的二十年中，基于甲基-TROSY的NMR实验已成为表征溶液中大蛋白和大分子机器动力学和功能机制的最重要手段之一。但是，大型蛋白质中甲基的化学位移分配仍然不容易，通常使用“分而治之”方法，依赖于骨架的实验，突变，基于结构的分配或这些的组合来进行。基于结构的甲基分配是一种新兴的策略，它减少了所需的时间和成本，并提供了一种与主链分配无关的方法。可用的基于结构的分配方案中的关键步骤之一是连接亮氨酸和缬氨酸残基的两个前手性甲基。以前，这是通过记录混合时间较短的NOESY光谱或通过比较NOESY光谱来实现的。在这里，我们提出了一种基于键合标量耦合转移的方法，即3D-HMBC-HMQC实验，以连接亮氨酸和缬氨酸的残基内甲基。结果表明，与仅使用NOESY光谱相比，HMBC-HMQC方法具有多个优势，因为观察到了独特的残留物内部交叉峰。此外，通过HMBC-HMQC实验可以轻松识别甲基-TROSY HMQC光谱中的重叠，从而消除了赋值中可能存在的歧义。结果表明，与仅使用NOESY光谱相比，HMBC-HMQC方法具有多个优势，因为观察到了独特的残留物内部交叉峰。此外，通过HMBC-HMQC实验可以轻松识别甲基-TROSY HMQC谱图中的重叠，从而消除了赋值中可能存在的歧义。结果表明，与仅使用NOESY光谱相比，HMBC-HMQC方法具有多个优势，因为观察到了独特的残留物内部交叉峰。此外，通过HMBC-HMQC实验可以轻松识别甲基-TROSY HMQC谱图中的重叠，从而消除了赋值中可能存在的歧义。