Groningen Biomolecular Sciences & Biotechnology Institute, University of Groningen, 9747 AG Groningen, The Netherlands.
Department of Industrial Engineering, University of Rome Tor Vergata, Via del Politecnico 1, 00133 Rome, Italy.
ACS Nano. 2023 Jul 25;17(14):13685-13699. doi: 10.1021/acsnano.3c02847. Epub 2023 Jul 17.
Nanopores are promising single-molecule tools for the electrical identification and sequencing of biomolecules. However, the characterization of proteins, especially in real-time and in complex biological samples, is complicated by the sheer variety of sizes and shapes in the proteome. Here, we introduce a large biological nanopore, YaxAB for folded protein analysis. The 15 nm -opening and a 3.5 nm -constriction describe a conical shape that allows the characterization of a wide range of proteins. Molecular dynamics showed proteins are captured by the electroosmotic flow, and the overall resistance is largely dominated by the narrow constriction region of the nanopore. Conveniently, proteins in the 35-125 kDa range remain trapped within the conical lumen of the nanopore for a time that can be tuned by the external bias. Contrary to cylindrical nanopores, in YaxAB, the current blockade decreases with the size of the trapped protein, as smaller proteins penetrate deeper into the constriction region than larger proteins do. These characteristics are especially useful for characterizing large proteins, as shown for pentameric C-reactive protein (125 kDa), a widely used health indicator, which showed a signal that could be identified in the background of other serum proteins.
纳米孔是一种有前途的单分子工具，可用于电识别和测序生物分子。然而，蛋白质的特性，特别是在实时和复杂的生物样本中，由于蛋白质组中大小和形状的多样性而变得复杂。在这里，我们引入了一种用于折叠蛋白质分析的大型生物纳米孔 YaxAB。15nm 的开口和 3.5nm 的收缩描述了一种锥形形状，允许对广泛的蛋白质进行特征描述。分子动力学表明，蛋白质被电渗流捕获，并且整体电阻主要由纳米孔的狭窄收缩区域主导。方便的是，35-125 kDa 范围内的蛋白质在纳米孔的锥形腔室内被捕获的时间可以通过外部偏置来调整。与圆柱形纳米孔不同，在 YaxAB 中，电流阻塞随着被捕获蛋白质的大小而减小，因为较小的蛋白质比较大的蛋白质更深入地穿透到收缩区域。这些特性对于表征大蛋白质特别有用，如五聚体 C 反应蛋白（125 kDa）所示，它是一种广泛使用的健康指标，其信号可以在其他血清蛋白的背景下识别。
