The design and preparation of high spatiotemporal resolution pore-forming proteins are crucial for achieving single-molecule analysis and sequencing of biomacromolecules. 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, herein, 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.
The related paper entitled “Single-Molecule Sensing inside Stereo- and Regio-Defined Hetero-Nanopores” has been published on Nature Nanotechnology on August 20, 2024 (Paper link: https://doi.org/10.1038/s41565-024-01721-2). Prof. Yi-Lun Ying is the co-corresponding author. PhD. student Wei Liu, Dr. Qiang Zhu and PhD. student Chao-Nan Yang are the co-first authors. Prof. Jing Ma and Professor Yi-Tao Long provided important guidance in all-atom molecular dynamics simulations and nanopore electrochemical measurements, respectively. This research was supported by the National Natural Science Foundation of China and the National Key Research and Development Program of China.
Fig. 1 Synthesis by Sensing strategy and discrimination of stereoisomers and diastereomers.
Fig. 2 In situ recording of single-molecule electrical trace to monitor the stepwise chemical modification process within protein nanopores.
Fig. 3 The asymmetric structural domains of the heteromeric protein nanopore characterized by cryo-electron microscopy and all-atom molecular dynamics simulations.
Fig. 4 Precise identification and label-free quantification of peptide stereoisomers and diastereomers inside the heteromeric protein nanopore.