Fluorescentproteins(FPs)withemissionwavelengthsinthefar-redandinfraredregionsofthespectrumprovidepowerfultoolsfordeep-tissueandsuper-resolutionimaging.Thedevelopmentofred-shiftedFPshasevokedwidespreadinterestandcontinuousengineeringefforts.Inthisarticle,basedonacomputationaldesignandgeneticcodeexpansion,wereportarationalapproachtosignificantlyexpandandred-shiftthechromophoreofgreenfluorescentprotein(GFP).WeappliedcomputationalcalculationstopredicttheexcitationandemissionwavelengthsofaFPchromophoreharboringunnaturalaminoacids(UAA)andidentifyinsilicoanappropriateUAA,2-amino-3-(6-hydroxynaphthalen-2-yl)propanoicacid(naphthol-Ala).OurmethodologyallowedustoformulateaGFPvariant(cpsfGFP-66-Naphthol-Ala)withred-shiftedabsorbanceandemissionspectralmaximaexceeding60and130nm,respectively,comparedtothoseofGFP.TheGFPchromophoreisformedthroughautocatalyticpost-translationalmodificationtogenerateaplanar4-(p-hydroxybenzylidene)-5-imidazolinonechromophore.WesolvedthecrystalstructureofcpsfGFP-66-naphthol-Alaat1.3■resolutionanddemonstratedtheformationofamuchlargerconjugatedn-systemwhenthephenolgroupisreplacedbynaphthol.Theseresultsexplainthesignificantred-shiftingoftheexcitationandemissionspectraofcpsfGFP-66-naphthol-Ala.
