Biosynthesis of a Highly Stable Coiled-Coil Protein Containing Hexafluoroleucine in an Engineered Bacterial Host
Incorporation of nonnatural amino acid residues allows engineering of proteins with novel chemical functionality and unusual physical properties. We have shown recently that coiled-coil proteins prepared in vivo can be stabilized significantly by replacement of leucine by trifluoroleucine (1). In the same series of experiments, however, we were unsuccessful in our attempts to incorporate the more highly fluorinated analogue hexafluoroleucine (2). We report here that modification of the leucyl-tRNA synthetase (LeuRS) activity of the host allows efficient incorporation of 2 into recombinant proteins prepared in Escherichia coli. Furthermore, the coiled-coil protein used to demonstrate incorporation of 2 exhibits enhanced stability in comparison to the same protein enriched in 1, possibly due to the increased hydrophobic character of the additional trifluoromethyl group in the protein core.