Instituto Cajal, IC-CSIC, Avda. Doctor Arce 37, E-28002 Madrid, Spain.
Nanoscale. 2018 Sep 13;10(35):16857-16867. doi: 10.1039/c8nr02785d.
Intrinsically disordered proteins (IDPs) lack a tertiary structure. Amyloidogenic IDPs (aIDPs) in particular have attracted great interest due to their implication in several devastating diseases as well as in critical biological functions. However, the conformational changes that trigger amyloid formation in aIDPs are largely unknown. aIDPs' conformational polymorphism at the monomer level encumbers their study using bulk techniques. Single-molecule techniques like atomic force microscopy-based single-molecule force spectroscopy represent a promising approach and a "carrier-guest" strategy, in which the protein of interest is mechanically protected, was developed to overcome the spurious signals from the noisy proximal region. However, since the carrier and single-molecule markers have similar mechanostabilities, their signals can intermingle in the force-extension recordings, making peak selection and analysis very laborious, cumbersome and prone to error for the non-expert. Here we have developed a new carrier, the c8C module from the CipC scaffoldin, with a higher mechanostability so that the signals from the protected protein will appear at the end of the recordings. This assures an accurate, more efficient and expert-independent analysis, simplifying both the selection and analysis of the single-molecule data. Furthermore, this modular design can be integrated into any SMFS polyprotein-based vector, thus constituting a useful utensil in the growing toolbox of protein nanomechanics.
无规卷曲蛋白质（IDPs）缺乏三级结构。特别是淀粉样变构 IDPs（aIDPs），由于其与多种破坏性疾病以及关键的生物学功能有关，引起了广泛关注。然而，触发 aIDPs 中淀粉样形成的构象变化在很大程度上仍是未知的。aIDPs 在单体水平上的构象多态性阻碍了使用体相技术对其进行研究。基于原子力显微镜的单分子力谱等单分子技术代表了一种很有前途的方法，并且开发了“载体-客体”策略，其中感兴趣的蛋白质在机械上受到保护，以克服来自嘈杂近侧区域的虚假信号。然而，由于载体和单分子标记具有相似的力学稳定性，它们的信号会在力-延伸记录中混合，使得峰选择和分析对于非专家来说非常费力、繁琐且容易出错。在这里，我们开发了一种新的载体，即 CipC 支架蛋白的 c8C 模块，其力学稳定性更高，从而使受保护的蛋白质的信号将出现在记录的末端。这确保了准确、更高效和无需专家干预的分析，简化了单分子数据的选择和分析。此外，这种模块化设计可以集成到任何基于 SMFS 多蛋白的载体中，因此构成了蛋白质纳米力学不断增长的工具包中的有用工具。
