NMR spectroscopy allows the study of biomolecules in close-to-native conditions. Structural information can be inferred from the NMR spectra when an assignment is available. Protein assignment is usually a time-consuming task, being specially challenging in the case of large, supramolecular systems. Here, we present an extension of existing state-of-the-art strategies for methyl group assignment that partially overcomes signal overlapping and other difficulties associated to isolated methyl groups. Our approach exploits the ability of proteins to populate two or more conformational states, allowing for unique NOE restraints in each protein conformer. The method is compatible with automated assignment algorithms, granting assignments beyond the limits of a single protein state. The approach also benefits from long-range structural restraints obtained from metal-induced pseudocontact shifts (PCS) and paramagnetic relaxation enhancements (PREs). We illustrate the method with the complete assignment of the 199 methyl groups of a MIL^proSV^proSAT methyl-labeled sample of the UDP-glucose pyrophosphorylase enzyme from Leishmania major (LmUGP). Protozoan parasites of the genus Leishmania causes Leishmaniasis, a neglected disease affecting over 12 million people worldwide. LmUGP is responsible for the de novo biosynthesis of uridine diphosphate-glucose, a precursor in the biosynthesis of the dense surface glycocalyx involved in parasite survival and infectivity. NMR experiments with LmUGP and related enzymes have the potential to unravel new insights in the host resistance mechanisms used by Leishmania major. Our efforts will help in the development of selective and efficient drugs against Leishmania.

核磁共振光谱允许在接近天然的条件下研究生物分子。当分配可用时，可以从 NMR 光谱推断结构信息。蛋白质分配通常是一项耗时的任务，在大型超分子系统的情况下尤其具有挑战性。在这里，我们提出了现有最先进的甲基分配策略的扩展，该策略部分克服了与孤立甲基相关的信号重叠和其他困难。我们的方法利用蛋白质填充两种或多种构象状态的能力，允许在每个蛋白质构象异构体中进行独特的 NOE 限制。该方法与自动分配算法兼容，授予超出单个蛋白质状态限制的分配。该方法还受益于从金属诱导的伪接触位移 (PCS) 和顺磁弛豫增强 (PRE) 获得的远程结构约束。我们通过完整分配 MIL 的 199 个甲基来说明该方法^proS V ^proS AT 甲基标记的来自利什曼原虫(LmUGP) 的 UDP-葡萄糖焦磷酸化酶样品。利什曼原虫属的原生动物寄生虫会导致利什曼病，这是一种被忽视的疾病，影响全世界超过 1200 万人。LmUGP 负责尿苷二磷酸 - 葡萄糖的从头生物合成，这是参与寄生虫存活和感染性的致密表面糖萼生物合成的前体。LmUGP 和相关酶的 NMR 实验有可能揭示利什曼原虫主要使用的宿主抗性机制的新见解。我们的努力将有助于开发针对利什曼原虫的选择性和有效药物。