Genetic Encoding of Photocaged Tyrosines with Improved Light-Activation Properties for the Optical Control of Protease Function

Abstract

We genetically encoded three new caged tyrosine analogues with improved photochemical properties by using an engineered pyrrolysyl-tRNA synthetase/tRNA_CUA pair in bacterial and mammalian cells. We applied the new tyrosine analogues to the photoregulation of firefly luciferase by caging its key tyrosine residue, Tyr340, and observed excellent off-to-on light switching. This reporter was then used to evaluate the activation rates of the different light-removable protecting groups in live cells. We identified the nitropiperonyl caging group as an excellent compromise between incorporation efficiency and photoactivation properties. To demonstrate applicability of the new caged tyrosines, an important proteolytic enzyme, tobacco etch virus (TEV) protease, was engineered for optical control. The ability to incorporate differently caged tyrosine analogues into proteins in live cells further expands the unnatural amino acid and optogenetic toolbox.

Keywords: genetic code expansion; protease; protein engineering; synthetic biology; unnatural amino acid.