{"title":"Single-molecule sensing inside stereo- and regio-defined hetero-nanopores","authors":"Wei Liu, Qiang Zhu, Chao-Nan Yang, Ying-Huan Fu, Ji-Chang Zhang, Meng-Yin Li, Zhong-Lin Yang, Kai-Li Xin, Jing Ma, Mathias Winterhalter, Yi-Lun Ying, Yi-Tao Long","doi":"10.1038/s41565-024-01721-2","DOIUrl":null,"url":null,"abstract":"<p>Heteromeric pore-forming proteins often contain recognition patterns or stereospecific selection filters. However, the construction of heteromeric pore-forming proteins for single-molecule sensing is challenging due to the uncontrollability of producing position isomers and difficulties in purification of regio-defined products. To overcome these preparation obstacles, we present an in situ strategy involving single-molecule chemical modification of a heptameric pore-forming protein to build a stereo- and regio-specific heteromeric nanopore (hetero-nanopore) with a subunit stoichiometric ratio of 3:4. The steric hindrance inherent in the homo-nanopore of K238C aerolysin directs the stereo- and regio-selective modification of maleimide derivatives. Our method utilizes real-time ionic current recording to facilitate controlled voltage manipulation for stoichiometric modification and position-based side-isomer removal. Single-molecule experiments and all-atom molecular dynamics simulations revealed that the hetero-nanopore features an asymmetric stereo- and regio-defined residue structure. The hetero-nanopore produced was characterized by mass spectrometry and single-particle cryogenic electron microscopy. In a proof-of-concept single-molecule sensing experiment, the hetero-nanopore exhibited 95% accuracy for label-free discrimination of four peptide stereoisomers with single-amino-acid structural and chiral differences in the mixtures. The customized hetero-nanopores could advance single-molecule sensing.</p>","PeriodicalId":18915,"journal":{"name":"Nature nanotechnology","volume":null,"pages":null},"PeriodicalIF":38.1000,"publicationDate":"2024-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Nature nanotechnology","FirstCategoryId":"88","ListUrlMain":"https://doi.org/10.1038/s41565-024-01721-2","RegionNum":1,"RegionCategory":"材料科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"MATERIALS SCIENCE, MULTIDISCIPLINARY","Score":null,"Total":0}
引用次数: 0
Abstract
Heteromeric pore-forming proteins often contain recognition patterns or stereospecific selection filters. However, the construction of heteromeric pore-forming proteins for single-molecule sensing is challenging due to the uncontrollability of producing position isomers and difficulties in purification of regio-defined products. To overcome these preparation obstacles, we present an in situ strategy involving single-molecule chemical modification of a heptameric pore-forming protein to build a stereo- and regio-specific heteromeric nanopore (hetero-nanopore) with a subunit stoichiometric ratio of 3:4. The steric hindrance inherent in the homo-nanopore of K238C aerolysin directs the stereo- and regio-selective modification of maleimide derivatives. Our method utilizes real-time ionic current recording to facilitate controlled voltage manipulation for stoichiometric modification and position-based side-isomer removal. Single-molecule experiments and all-atom molecular dynamics simulations revealed that the hetero-nanopore features an asymmetric stereo- and regio-defined residue structure. The hetero-nanopore produced was characterized by mass spectrometry and single-particle cryogenic electron microscopy. In a proof-of-concept single-molecule sensing experiment, the hetero-nanopore exhibited 95% accuracy for label-free discrimination of four peptide stereoisomers with single-amino-acid structural and chiral differences in the mixtures. The customized hetero-nanopores could advance single-molecule sensing.
期刊介绍:
Nature Nanotechnology is a prestigious journal that publishes high-quality papers in various areas of nanoscience and nanotechnology. The journal focuses on the design, characterization, and production of structures, devices, and systems that manipulate and control materials at atomic, molecular, and macromolecular scales. It encompasses both bottom-up and top-down approaches, as well as their combinations.
Furthermore, Nature Nanotechnology fosters the exchange of ideas among researchers from diverse disciplines such as chemistry, physics, material science, biomedical research, engineering, and more. It promotes collaboration at the forefront of this multidisciplinary field. The journal covers a wide range of topics, from fundamental research in physics, chemistry, and biology, including computational work and simulations, to the development of innovative devices and technologies for various industrial sectors such as information technology, medicine, manufacturing, high-performance materials, energy, and environmental technologies. It includes coverage of organic, inorganic, and hybrid materials.