Genetic Encoding of 3-Cyano-Tyrosine and Its Use in Controlling the Chromophore Isomeric State of the Fluorescent Protein mKate

Switchable β-barrel-type fluorescent proteins are essential genetically encoded probes for super-resolution imaging. The space required for chromophore cis–trans isomerisation can also provide an opportunity to introduce bulkier chemistry at the 3-position of the phenolic ring. Here, we report, to our knowledge, the first successful genetic encoding of 3-cyano-L-tyrosine (3CNY) into a protein. Using genetic code expansion, the cyano-containing tyrosine derivative is incorporated directly into the chromophore of mKate, a pH-dependent switchable red fluorescent protein. While mKate adopts a fluorescent phenolate cis-state chromophore at physiological pH, substituting the native tyrosine with 3CNY yields a functional protein exhibiting hypsochromically shifted spectral properties. Time-dependent density functional theory (TD-DFT) calculations indicate that 3CNY incorporation results in a trans state at pH 8 but, unlike mKate, is fluorescent. The electron-withdrawing cyano group potentially perturbs conjugation across the chromophore, thus lowering the barrier to cis–trans isomerisation. The trans form may also be stabilised by hydrogen bonds from the cyano group to the rest of the protein. Overall, the introduction of a genetically encoded 3-CNY tyrosine analogue into a fluorescent protein chromophore expands our mechanistic understanding and enables the incorporation of a new chemical tag directly into the chromophore.