Controlled Genetic Encoding of Unnatural Amino Acids in a Protein Nanopore

• Published in: Angewandte Chemie International Edition 2023-07, Vol.62 (29), p.e202300582-n/a
• Main Authors: Wu, Xue‐Yuan, Li, Meng‐Yin, Yang, Shao‐Jun, Jiang, Jie, Ying, Yi‐Lun, Chen, Peng R., Long, Yi‐Tao
• Format: Article
• Language: English
• Subjects: Aerolysin; Amino acids; Chemoreception; Genetic code; Genetic Code Expansion; Hydrophobicity; Molecular dynamics; Nanopore; Peptide Sensing; Peptides; Protein engineering; Proteins; Structure-function relationships; tRNA; Unnatural Amino Acid
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.202300582

Conventional protein engineering methods for modifying protein nanopores are typically limited to 20 natural amino acids, which restrict the diversity of the nanopores in structure and function. To enrich the chemical environment inside the nanopore, we employed the genetic code expansion (GCE) technique to site‐specifically incorporate the unnatural amino acid (UAA) into the sensing region of aerolysin nanopores. This approach leveraged the efficient pyrrolysine‐based aminoacyl‐tRNA synthetase‐tRNA pair for a high yield of pore‐forming protein. Both molecular dynamics (MD) simulations and single‐molecule sensing experiments demonstrated that the conformation of UAA residues provided a favorable geometric orientation for the interactions of target molecules and the pore. This rationally designed chemical environment enabled the direct discrimination of multiple peptides containing hydrophobic amino acids. Our work provides a new framework for endowing nanopores with unique sensing properties that are difficult to achieve using classical protein engineering approaches. We present the application of the genetic code expansion technique to achieve the site‐specific modification of the sensing region of a nanopore. The rationally designed conformation of unnatural amino acid (UAA) residues provides a favorable geometric orientation for the interactions of peptides and pore. The chemical environment of the sensing region facilitates the direct discrimination of the mixtures of peptides containing hydrophobic amino acids.