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Generating Permissive Site‐specific Unnatural Amino Acid Synthetases
Author(s) -
Mehl Ryan A,
MiyakeStoner Shigeki J,
Refakis Christian A.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.838.7
Subject(s) - rss , amino acid , computational biology , ranking (information retrieval) , computer science , substrate (aquarium) , transfer rna , chemistry , biochemistry , biology , artificial intelligence , gene , rna , ecology , operating system
Genetically incorporated unnatural amino acid (UAA) technologies are powerful tools that are greatly enhancing our ability to study and engineer biological systems. To incorporate UAAs into protein, the substrate recognition properties of an aminoacyl‐tRNA synthetase (RS) must be modified. Protocols to do so are technically simple but require time and optimization, which has significantly limited the accessibly of this important technology. At present, engineered RSs are evaluated on their efficiency and fidelity. We propose that a third parameter of substrate recognition – permissivity – is equally important. Permissive RSs, whose relaxed substrate recognition properties allows them to incorporate multiple unnatural amino acids (but not natural amino acids), would eliminate the need to generate new RSs for many new unnatural amino acids. Methods for quickly and easily assessing the permissivity of existing RSs and for generating permissive RSs are presented. We determined the degree of permissivity of two UAA‐RSs for a family of structurally related fluorinated UAAs (19F‐UAAs). We then increased the permissivity of the initial RSs to allow for incorporation of the family of 19F‐UAAs. We expect that results of this work will increase the accessibility of UAA technology and the use of new UAAs in proteins. Supported by F&M funds, NSF‐MCB‐0448297; Research Corporation (CC6364); and ACS‐PRF (42214‐GB4).