RESUMEN
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.
Asunto(s)
Hidrozoos/genética , Hidrozoos/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Animales , Técnicas Biosensibles , Color , Cristalografía por Rayos X , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hidrozoos/química , Proteínas Luminiscentes/química , Modelos Moleculares , Imagen Óptica , Filogenia , Electricidad EstáticaRESUMEN
mNeonGreen, an engineered green fluorescent protein (GFP) derived from lancelet, is one of the most brightly fluorescent homologs of Aequorea victoria jellyfish GFP (avGFP) yet reported. In this work, we investigated whether this bright fluorescence might be retained in homologs of mNeonGreen with modified chromophore structures and altered fluorescent hues. We found mNeonGreen to be generally less tolerant than avGFP to chromophore modification by substitution of the key chromophore-forming tyrosine residue with other aromatic amino acids. However, we were ultimately successful in creating a variant, designated as NeonCyan1, with a tryptophan-derived cyan fluorescent protein (CFP)-type chromophore, and two additional mutants with distinct spectral hues. Structural, computational, and photophysical characterization of NeonCyan1 and its variants provided insight into the factors that control the fluorescence emission color. Though not recommended as replacements for contemporary CFP variants, we demonstrate that NeonCyan1 variants are potentially suitable for live cell imaging applications.