Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, Groningen 9747AG, Netherlands.
ACS Nano. 2022 May 24;16(5):7258-7268. doi: 10.1021/acsnano.1c11455. Epub 2022 Mar 18.
Biological nanopores are emerging as sensitive single-molecule sensors for proteins and peptides. The heterogeneous charge of a polypeptide chain, however, can complicate or prevent the capture and translocation of peptides and unfolded proteins across nanopores. Here, we show that two β-barrel nanopores, aerolysin and cytotoxin K, cannot efficiently detect proteinogenic peptides from a trypsinated protein under a wide range of conditions. However, the introduction of an acidic-aromatic sensing region in the β-barrel dramatically increased the dwell time and the discrimination of peptides in the nanopore at acidic pH. Surprisingly, despite the fact that the two β-barrel nanopores have a similar diameter and an acidic-aromatic construction, their capture mechanisms differ. The electro-osmotic flow played a dominant role for aerolysin, while the electrophoretic force dominated for cytotoxin K. Nonetheless, both β-barrel nanopores allowed the detection of mixtures of trypsinated peptides, with aerolysin nanopores showing a better resolution for larger peptides and cytotoxin K showing a better resolution for shorter peptides. Therefore, this work provides a generic strategy for modifying nanopores for peptide detection that will be most likely be applicable to other nanopore-forming toxins.
生物纳米孔作为敏感的单分子蛋白质和肽传感器正在兴起。然而，多肽链的不均匀电荷会使肽和未折叠蛋白质在纳米孔中的捕获和转运复杂化或受阻。在这里，我们表明，在广泛的条件下，两种β-桶纳米孔（ Aerolysin 和 Cytotoxin K ）不能有效地从胰蛋白酶处理的蛋白质中检测到蛋白质肽。然而，在β-桶中引入酸性芳香感应区可显著增加酸性 pH 值下肽在纳米孔中的停留时间和区分度。令人惊讶的是，尽管这两种β-桶纳米孔具有相似的直径和酸性芳香结构，但它们的捕获机制不同。电渗流对 Aerolysin 起主导作用，而电泳力对 Cytotoxin K 起主导作用。尽管如此，两种β-桶纳米孔都可以检测到胰蛋白酶处理的肽混合物，Aerolysin 纳米孔对较大的肽具有更好的分辨率，而 Cytotoxin K 对较短的肽具有更好的分辨率。因此，这项工作为修饰用于肽检测的纳米孔提供了一种通用策略，该策略很可能适用于其他形成纳米孔的毒素。
