Genetic incorporation of unnatural amino acids into proteins

Abstract

The introduction of unnatural amino acids with novel properties into proteins is potentially a useful tool for understanding and manipulating their functions. The development of an in vitro method for the incorporation of a fluorescent amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco), into proteins is described here. An S30 extract was produced from E. coli culture containing a plasmid that encodes an evolved aminoacyl-tRNA synthetase specific for Hco (CouRS) and a suppressor tRNA (Sup-tRNA). Additionally, total tRNA, including the Sup-tRNA, and CouRS were expressed separately and purified. Expression of green fluorescent protein, Sortase and peptidyl prolyl isomerase B genes containing amber codons using the S30 extract, and in the presence of Hco yielded both full-length Hco-containing proteins and the truncated versions. Confirmation of insertion of Hco was performed by fluorescent analysis of proteins after denaturing SDS-PAGE electrophoresis. Whole protein mass spectrometry also showed that the experimentally determined mass of Sortase-Hco matched the mass calculated from its amino acid sequence. Tryptic digest mass spectrometry of Sortase-Hco confirmed the presence of the Hco-containing peptide, while another peptide detected after the amber codon in the Sortase sequence further confirmed the expression of full-length Sortase-Hco protein. The incorporation efficiency of Hco was determined to be approximately forty percent. Insertion of Hco was used to test whether the naturally evolved turnover rate of bacterial phosphotriesterases (PTEs) could be improved through the incorporation of unnatural amino acids and also to probe the role ofperipheral active site residues in non-chemical steps (substrate binding and product release) of the catalytic cycle. The naturally occurring tyrosine amino acid at position 309 was replaced with Hco and L-(7-methylcoumarin-4-yl)ethylglycine (Mco). Kinetic analysis suggests that the 7-hydroxyl group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product release step of paraoxon hydrolysis as a result of its electrostatic repulsion of the negatively charged 4-nitrophenolate product. The 8-fold improvement of this already highly efficient catalyst through a single rationally designed unnatural mutant is in stark contrast to the difficulty in improving this native activity through screening hundreds of thousands of natural variants, demonstrating that designer amino acids provide easy access to new sequence and functional space in the engineering and evolution of existing enzyme functions. Another application of Hco was to investigate the binding of small inhibitors to an enzyme target in a drug discovery project. Development of enzyme inhibitors requires an activity assay for the identification of hits and lead compounds. To determine dissociation constants in a straightforward manner, the use of site-specific tagging of the target protein was explored. Hco was site-specifically incorporated in the target protein West Nile virus NS2B-NS3 protease and the fluorescence of the Hco tagged samples proved to be exquisitely sensitive to the presence of inhibitors and small ligand molecules if they bind in the vicinity of the Hco residue. No significant change in fluorescence was observed when the ligand binding site was far from the Hco residue. Hco-tagged proteins thus combine outstanding sensitivity with accurate information on the site ofbinding, making Hco labelling an attractive tool in drug discovery. Show more