Data_Sheet_1_NMR Structure and Dynamics of TonB Investigated by Scar-Less Segmental Isotopic Labeling Using a Salt-Inducible Split Intein.pdf

The growing understanding of partially unfolded proteins increasingly points to their biological relevance in allosteric regulation, complex formation, and protein design. However, the structural characterization of disordered proteins remains challenging. NMR methods can access both the dynamics and structures of such proteins, yet suffering from a high degeneracy of NMR signals. Here, we overcame this bottleneck utilizing a salt-inducible split intein to produce segmentally isotope-labeled samples with the native sequence, including the ligation junction. With this technique, we investigated the NMR structure and conformational dynamics of TonB from Helicobacter pylori in the presence of a proline-rich low complexity region. Spin relaxation experiments suggest that the several nano-second time scale dynamics of the C-terminal domain (CTD) is almost independent of the faster pico-to-nanosecond dynamics of the low complexity central region (LCCR). Our results demonstrate the utility of segmental isotopic labeling for proteins with heterogenous dynamics such as TonB and could advance NMR studies of other partially unfolded proteins.

对部分折叠蛋白质的理解日益加深，逐渐揭示了其在别构调节、复杂体形成和蛋白质设计中的生物学重要性。然而，无序蛋白质的结构表征仍然颇具挑战。核磁共振技术能够探测此类蛋白质的动态和结构，但仍受限于核磁共振信号的强歧义性。在本研究中，我们利用盐诱导的分裂连接蛋白，成功克服了这一瓶颈，生产出包含天然序列及连接接点的分段同位素标记样本。通过这一技术，我们探究了幽门螺杆菌中的TonB蛋白在富含脯氨酸的低复杂度区域存在时的核磁共振结构和构象动态。自旋弛豫实验表明，C端结构域（CTD）在数纳秒时间尺度上的动态几乎独立于低复杂度中央区域（LCCR）在皮秒至纳秒时间尺度上的快速动态。我们的研究结果证明了分段同位素标记对于如TonB蛋白这类动态异质蛋白质的实用性，并可能推动对其他部分折叠蛋白质的核磁共振研究的进展。