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Abstract
Recent advances have fundamentally changed the ways in which synthetic amino acids are incorporated into proteins, enabling their efficient and multiple-site incorporation, in addition to the 20 canonical amino acids. This development provides opportunities for fresh approaches toward addressing fundamental problems in bioengineering. In the present study, we showed that the structural stability of proteins can be enhanced by integrating bulky halogenated amino acids at multiple selected sites. Glutathione S-transferase was thus stabilized significantly (by 5.2 and 5.6 kcal/mol) with 3-chloro- and 3-bromo-l-tyrosines, respectively, incorporated at seven selected sites. X-ray crystallographic analyses revealed that the bulky halogen moieties filled internal spaces within the molecules, and formed non-canonical stabilizing interactions with the neighboring residues. This new mechanism for protein stabilization is quite simple and applicable to a wide range of proteins, as demonstrated by the rapid stabilization of the industrially relevant azoreductase.
Highlights
Recent advances have fundamentally changed the ways in which synthetic amino acids are incorporated into proteins, enabling their efficient and multiple-site incorporation, in addition to the 20 canonical amino acids
Release factor 1 (RF-1) has been eliminated from Escherichia coli, and the in vivo meaning of UAG has been redefined from a stop codon to a sense codon specific for synthetic amino acids[5,6,7,8]
We previously synthesized an iodinated variant (7iGST) of eukaryotic glutathione S-transferase (GST), a homodimeric detoxification enzyme, in which seven specific residues of the 15 tyrosines per monomer were replaced by 3-iodo-L-tyrosines[6]
Summary
Kazumasa Ohtake[1,2], Atsushi Yamaguchi[1,2], Takahito Mukai1,2*, Hiroki Kashimura[3], Nobutaka Hirano[3], Mitsuru Haruki[3], Sosuke Kohashi[3], Kenji Yamagishi[3], Kazutaka Murayama[4], Yuri Tomabechi[1,2], Takashi Itagaki[2], Ryogo Akasaka[1,2], Masahito Kawazoe[2,5], Chie Takemoto[1,2], Mikako Shirouzu[1,2], Shigeyuki Yokoyama2,5 & Kensaku Sakamoto[1,2]
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