Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
J Am Chem Soc. 2011 Mar 30;133(12):4175-7. doi: 10.1021/ja109469c. Epub 2011 Mar 1.
Multidomain proteins constitute a large part of prokaryotic and eukaryotic proteomes and play fundamental roles in various physiological processes. However, their structural characterization is challenging because of their large size and intrinsic flexibility. We show here that motional-filtered high-resolution solid-state NMR (ssNMR) experiments allow for the observation and structural analysis of very large multidomain membrane proteins that are characterized by different motional time scales. This approach was used to probe the folding of the 790-residue membrane protein BamA, which is the core component of the Escherichia coli outer membrane protein assembly machinery. A combination of dipolar- and scalar-based two-dimensional ssNMR experiments applied to two uniformly (13)C,(15)N-labeled BamA variants revealed characteristic secondary structure elements and distinct dynamics within the BamA transmembrane protein segment and the periplasmic POTRA domains. This approach hence provides a general strategy for collecting atomic-scale structural information on multidomain (membrane) proteins in a native-like environment.
多结构域蛋白构成原核生物和真核生物蛋白质组的很大一部分，在各种生理过程中发挥着基本作用。然而，由于它们的体积大和固有灵活性，它们的结构表征具有挑战性。我们在这里展示，运动过滤的高分辨率固态 NMR（ssNMR）实验允许观察和结构分析具有不同运动时间尺度的非常大的多结构域膜蛋白。该方法用于探测 790 个残基的膜蛋白 BamA 的折叠，BamA 是大肠杆菌外膜蛋白组装机制的核心组件。应用于两种均（13）C、（15）N 标记的 BamA 变体的基于偶极子和标量的二维 ssNMR 实验揭示了 BamA 跨膜蛋白段和周质 POTRA 结构域内的特征二级结构元件和不同的动力学。因此，该方法为在类似天然环境中收集多结构域（膜）蛋白的原子尺度结构信息提供了一种通用策略。
