title: The photophysics of green fluorescent protein: influence of the key amino acids at positions 65, 203, and 222.
abstract: The three amino acids S65, T203, and E222 crucially determine the photophysical behavior of wild-type green fluorescent protein. We investigate the impact of four point mutations at these positions and their respective combinations on green fluorescent protein's photophysics using absorption spectroscopy, as well as steady-state and time-resolved fluorescence spectroscopy. Our results highlight the influence of the protein's hydrogen-bonding network on the equilibrium between the different chromophore states and on the efficiency of the excited-state proton transfer. The mutagenic approach allows us to separate different mechanisms responsible for fluorescence quenching, some of which were previously discussed theoretically. Our results will be useful for the development of new strategies for the generation of autofluorescent proteins with specific photophysical properties. One example presented here is a variant exhibiting uncommon blue fluorescence.
authors.affiliation: Department Chemie and Center for Nanoscience, LMU Munich, Munich, Germany.
authors.forename: Gregor
authors.initials: G
authors.lastname: Jung
authors.forename: Jens
authors.initials: J
authors.lastname: Wiehler
authors.forename: Andreas
authors.initials: A
authors.lastname: Zumbusch
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids / Q000032:analysis / Q000737:chemistry* / Q000528:radiation effects*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects*; D008027:Light; D016297:Mutagenesis, Site-Directed; D018462:Photobiology / Q000379:methods; D010777:Photochemistry / Q000379:methods; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
chemical_list: D000596:Amino Acids; D011994:Recombinant Proteins; C485192:green fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins