Stevenson, C.; Mclarnon, J.; Harnedy, J.; Elsherbeni, S.; Saha, D.; Langbein, W.; Borri, P.; Platts, J.; Morrill, L.; Jones, D.

Switchable beta 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 amino acid was incorporated directly into the chromophore of mKate, a pH-dependent switchable red fluorescent protein. In mKate, the chromophore adopts a fluorescent phenolate cis state at physiological pH, transitioning to a phenolic trans state under acidic conditions. Substitution of 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. Unlike mKate, the trans state is fluorescent. In contrast, incorporation of 3-chloro-L-tyrosine (3ClY) preserves the preference for the cis phenolate state. Molecular modelling suggests that the cyano group can form stabilising hydrogen bonds with residues S143 and S158, promoting the trans configuration. DFT analysis further indicates that the electron-withdrawing cyano group perturbs conjugation across the chromophore, potentially lowering the barrier to cis-trans isomerisation. Conversely, wild-type and 3ClY variants maintain polarised HOMO and LUMO distributions in the cis state, supporting stronger conjugation and a reduced HOMO-LUMO gap. Overall, the introduction of a genetically encoded 3-CNY tyrosine analogue into a fluorescent protein chromophore expands our mechanistic understanding and enables incorporation of a new chemical tag directly into the chromophore.