Biosynthesis of a Highly Stable Coiled-Coil Protein Containing Hexafluoroleucine in an Engineered Bacterial Host

Abstract

Incorporation of nonnatural amino acid residues allows engineering \nof proteins with novel chemical functionality and unusual \nphysical properties. We have shown recently that coiled-coil \nproteins prepared in vivo can be stabilized significantly by \nreplacement of leucine by trifluoroleucine (1). In the same series \nof experiments, however, we were unsuccessful in our attempts \nto incorporate the more highly fluorinated analogue hexafluoroleucine \n(2). We report here that modification of the leucyl-tRNA \nsynthetase (LeuRS) activity of the host allows efficient incorporation \nof 2 into recombinant proteins prepared in Escherichia coli. \nFurthermore, the coiled-coil protein used to demonstrate incorporation \nof 2 exhibits enhanced stability in comparison to the \nsame protein enriched in 1, possibly due to the increased \nhydrophobic character of the additional trifluoromethyl group in \nthe protein core.