Generating Permissive Site-Specific Unnatural Amino Acid Synthetases for In Vivo Production of Proteins Containing Useful Chemical Functionality
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 allow 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 for a variety of UAA containing useful chemical functionality will be presented. We determined the degree of permissivity of many existing UAA-RSs for families of structurally related UAAs. We then increased the permissivity of the initial RSs to allow for the incorporation of the families of 19F-UAAs and photocrosslinking UAAs. We expect that the results of this work will increase the accessibility of UAA technology and the use of new UAAs in proteins.