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Proximity‐Enabled Protein Crosslinking through Genetically Encoding Haloalkane Unnatural Amino Acids
Author(s) -
Xiang Zheng,
Lacey Vanessa K.,
Ren Haiyan,
Xu Jing,
Burban David J.,
Jennings Patricia A.,
Wang Lei
Publication year - 2014
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.201308794
Subject(s) - covalent bond , chemistry , amino acid , cysteine , protein engineering , aminoacyl trna synthetase , thermostability , protein design , protein structure , stereochemistry , biochemistry , transfer rna , organic chemistry , enzyme , rna , gene
The selective generation of covalent bonds between and within proteins would provide new avenues for studying protein function and engineering proteins with new properties. New covalent bonds were genetically introduced into proteins by enabling an unnatural amino acid (Uaa) to selectively react with a proximal natural residue. This proximity‐enabled bioreactivity was expanded to a series of haloalkane Uaas. Orthogonal tRNA/synthetase pairs were evolved to incorporate these Uaas, which only form a covalent thioether bond with cysteine when positioned in close proximity. By using the Uaa and cysteine, spontaneous covalent bond formation was demonstrated between an affibody and its substrate Z protein, thereby leading to irreversible binding, and within the affibody to increase its thermostability. This strategy of proximity‐enabled protein crosslinking (PEPC) may be generally expanded to target different natural amino acids, thus providing diversity and flexibility in covalent bond formation for protein research and protein engineering.