内禀无序蛋白质（IDPs）中富集了很多脯氨酸残基，它们可以形成特定的局部次级结构元素，被称为PPII螺旋，其特点是紧密包装程度较小。脯氨酸经常被认为促进无序，但它可以参与与芳香侧链之间的特定π·CH相互作用，从而减少多肽的构象柔性。差异的局部动力学对于预形成的结构元素的稳定性以及长程相互作用的建立起到关键作用。进行核磁共振（NMR）实验以探测脯氨酸环系统的动力学是非常理想的。在这里，我们提出了一种基于13C信号检测的脯氨酸甲基CH2基团之间偶极偶极交叉弛豫（CCR）速率的脉冲方案。应用13C-CON检测策略提供了精细的谱分辨率，即使在接近生理条件下，也能适用于高分子量的IDPs。该脉冲方案通过应用于一个包含220个氨基酸的蛋白质Osteopontin的实例进行了说明，Osteopontin是一种参与炎症和癌症进展的细胞外细胞因子，所述实例中的蛋白质构造发生了三个脯氨酸-芳香序列区域的突变。 版权所有 © 2023 Elsevier Inc. 发表

Intrinsically disordered proteins (IDPs) are significantly enriched in proline residues, which can populate specific local secondary structural elements called PPII helices, characterized by small packing densities. Proline is often thought to promote disorder, but it can participate in specific π·CH interactions with aromatic side chains resulting in reduced conformational flexibilities of the polypeptide. Differential local motional dynamics are relevant for the stabilization of preformed structural elements and can serve as nucleation sites for the establishment of long-range interactions. NMR experiments to probe the dynamics of proline ring systems would thus be highly desirable. Here we present a pulse scheme based on 13C detection to quantify dipole-dipole cross-correlated relaxation (CCR) rates at methylene CH2 groups in proline residues. Applying 13C-CON detection strategy provides exquisite spectral resolution allowing applications also to high molecular weight IDPs even in conditions approaching the physiological ones. The pulse scheme is illustrated with an application to the 220 amino acids long protein Osteopontin, an extracellular cytokine involved in inflammation and cancer progression, and a construct in which three proline-aromatic sequence patches have been mutated.Copyright © 2023. Published by Elsevier Inc.