RESUMO
Using mRNA sequencing and de novo transcriptome assembly, we identified, cloned, and characterized 9 previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from A. victoria green fluorescent protein (avGFP). Among these FPs are the brightest green fluorescent protein (GFP) homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including 2 that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.
Assuntos
Hidrozoários/genética , Hidrozoários/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Animais , Técnicas Biossensoriais , Cor , Cristalografia por Raios X , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hidrozoários/química , Proteínas Luminescentes/química , Modelos Moleculares , Imagem Óptica , Filogenia , Eletricidade EstáticaRESUMO
We report the engineering of mScarlet, a truly monomeric red fluorescent protein with record brightness, quantum yield (70%) and fluorescence lifetime (3.9 ns). We developed mScarlet starting with a consensus synthetic template and using improved spectroscopic screening techniques; mScarlet's crystal structure reveals a planar and rigidified chromophore. mScarlet outperforms existing red fluorescent proteins as a fusion tag, and it is especially useful as a Förster resonance energy transfer (FRET) acceptor in ratiometric imaging.