StayRose: a photostable StayGold derivative red-shifted by genetic code expansion

Photobleaching of fluorescent proteins often limits the acquisition of high-quality images in microscopy. StayGold, a novel dimeric green fluorescent protein recently monomerised through sequence engineering, addresses this challenge with its high photostability. There is now focus on producing different colour StayGold derivatives to facilitate concurrent tagging of multiple targets. The unnatural amino acid 3-aminotyrosine has previously been shown to red-shift super folder GFP upon incorporation into its chromophore via genetic code expansion. Here we apply the same strategy to red-shift StayGold through substitution of Tyrosine-58 with 3-aminotyrosine. The resultant red fluorescent protein, StayRose, shows 530 nm excitation and 588 nm emission peaks, shifting from the 497 nm and 504 nm excitation and emission peaks of StayGold. StayRose also retains the favourable photostability of StayGold and can be similarly monomerised using mutations at the dimer interface. A high-resolution crystal structure of StayRose confirms the modified structure of the amino-chromophore within an unperturbed 3D fold. Although reliant on genetic code expansion, StayRose provides an important step towards developing red-shifted StayGold derivatives.