The development of methyl transverse relaxation optimized spectroscopy has greatly facilitated the study of macromolecular assemblies by solution NMR spectroscopy. However, limited sample solubility and stability has hindered application of this technique to ongoing studies of complexes formed on membranes by the neuronal SNAREs that mediate neurotransmitter release and synaptotagmin-1, the Ca²⁺ sensor that triggers release. Since the ¹H NMR signal of a ^tBu group attached to a large protein or complex can be observed with high sensitivity if the group retains high mobility, we have explored the use of this strategy to analyze presynaptic complexes involved in neurotransmitter release. For this purpose, we attached ^tBu groups at single cysteines of fragments of synaptotagmin-1, complexin-1 and the neuronal SNAREs by reaction with 5-(tert-butyldisulfaneyl)-2-nitrobenzoic acid (BDSNB), ^tBu iodoacetamide or ^tBu acrylate. The ^tBu resonances of the tagged proteins were generally sharp and intense, although ^tBu groups attached with BDSNB had a tendency to exhibit somewhat broader resonances that likely result because of the shorter linkage between the ^tBu and the tagged cysteine. Incorporation of the tagged proteins into complexes on nanodiscs led to severe broadening of the ^tBu resonances in some cases. However, sharp ^tBu resonances could readily be observed for some complexes of more than 200 kDa at low micromolar concentrations. Our results show that tagging of proteins with ^tBu groups provides a powerful approach to study large biomolecular assemblies of limited stability and/or solubility that may be applicable even at nanomolar concentrations.

甲基横向弛豫优化光谱的发展极大地促进了溶液核磁共振光谱对大分子组装体的研究。然而，有限的样品溶解度和稳定性阻碍了该技术在膜上由介导神经递质释放的神经元 SNARE 和触发释放的 Ca ^{2+传感器突触结合蛋白 1 形成的复合物的持续研究中的应用。}由于连接到大蛋白质或复合物的^{t Bu 基团的1} H NMR 信号可以以高灵敏度观察到，如果该基团保持高迁移率，我们已经探索了使用这种策略来分析参与神经递质释放的突触前复合物。为此，我们附上^了通过与 5-(叔丁基二硫烷基)-2-硝基苯甲酸 (BDSNB)、^t Bu 碘乙酰胺或^t Bu 丙烯酸酯反应，突触结合蛋白-1、络合蛋白-1 和神经元 SNARE 片段的单个半胱氨酸上的 Bu 基团。标记蛋白质的^t Bu 共振通常是尖锐和强烈的，尽管与 BDSNB 连接的^t Bu 基团倾向于表现出更广泛的共振，这可能是由于^t Bu 和标记的半胱氨酸之间的连接较短而导致的。在某些情况下，将标记的蛋白质结合到纳米盘上的复合物中会导致^t Bu 共振的严重展宽。然而，尖锐的^吨对于一些超过 200 kDa 在低微摩尔浓度下的复合物，可以很容易地观察到 Bu 共振。我们的结果表明，用^t Bu 基团标记蛋白质提供了一种强大的方法来研究具有有限稳定性和/或溶解度的大型生物分子组装体，即使在纳摩尔浓度下也可能适用。