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

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.