Controlled Genetic Encoding of Unnatural Amino Acids in a Protein Nanopore

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) tech...

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Bibliographic Details
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
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Summary: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.
ISSN:1433-7851
1521-3773
DOI:10.1002/anie.202300582