Department of Chemistry, Western Washington University, Bellingham, Washington, United States of America.
Department of Biology, Western Washington University, Bellingham, Washington, United States of America.
PLoS One. 2021 Oct 28;16(10):e0258531. doi: 10.1371/journal.pone.0258531. eCollection 2021.
A significant number of proteins possess sizable intrinsically disordered regions (IDRs). Due to the dynamic nature of IDRs, NMR spectroscopy is often the tool of choice for characterizing these segments. However, the application of NMR to IDRs is often hindered by their instability, spectral overlap and resonance assignment difficulties. Notably, these challenges increase considerably with the size of the IDR. In response to these issues, here we report the use of sortase-mediated ligation (SML) for segmental isotopic labeling of IDR-containing samples. Specifically, we have developed a ligation strategy involving a key segment of the large IDR and adjacent folded headpiece domain comprising the C-terminus of A. thaliana villin 4 (AtVLN4). This procedure significantly reduces the complexity of NMR spectra and enables group identification of signals arising from the labeled IDR fragment, a process we refer to as segmental assignment. The validity of our segmental assignment approach is corroborated by backbone residue-specific assignment of the IDR using a minimal set of standard heteronuclear NMR methods. Using segmental assignment, we further demonstrate that the IDR region adjacent to the headpiece exhibits nonuniform spectral alterations in response to temperature. Subsequent residue-specific characterization revealed two segments within the IDR that responded to temperature in markedly different ways. Overall, this study represents an important step toward the selective labeling and probing of target segments within much larger IDR contexts. Additionally, the approach described offers significant savings in NMR recording time, a valuable advantage for the study of unstable IDRs, their binding interfaces, and functional mechanisms.
大量蛋白质拥有相当大的无规则区域（IDR）。由于 IDR 的动态性质，NMR 光谱通常是用于描述这些片段的首选工具。然而，NMR 在 IDR 中的应用常常受到其不稳定性、光谱重叠和共振分配困难的限制。值得注意的是，这些挑战随着 IDR 的大小而大大增加。针对这些问题，我们在这里报告了使用 sortase 介导的连接（SML）对含有 IDR 的样品进行分段同位素标记的方法。具体来说，我们开发了一种涉及 IDR 中关键片段和相邻折叠头片域的连接策略，该头片域包含拟南芥卷曲蛋白 4（AtVLN4）的 C 末端。该方法显著降低了 NMR 光谱的复杂性，并能够对标记的 IDR 片段的信号进行分组识别，我们称之为分段分配。通过使用一组最小的标准异核 NMR 方法对 IDR 进行残基特异性分配，验证了我们的分段分配方法的有效性。使用分段分配，我们进一步表明，头片附近的 IDR 区域对温度表现出非均匀的光谱变化。随后的残基特异性表征揭示了 IDR 内的两个片段以明显不同的方式对温度做出响应。总体而言，这项研究代表了在更大的 IDR 背景下选择性标记和探测目标片段的重要一步。此外，所描述的方法在 NMR 记录时间方面具有显著的节省，这对于不稳定的 IDR、它们的结合界面和功能机制的研究是一个有价值的优势。
