title: Hidden electronic excited state of enhanced green fluorescent protein.
abstract: Precise two-photon absorption spectra of the green fluorescent protein (GFP) and the mutants sapphire-GFP (T203I) and enhanced GFP (S65T/F64L), as well as a model compound for the chromophore, 4'-hydroxybenzylidene-2,3-dimethylimidazolinone (HBDI) were measured by multiplex two-photon absorption spectroscopy. The observed TPA bands of the anionic forms of enhanced GFP and HBDI were significantly shifted to the higher energy compared with the lowest-energy bands in one-photon absorption spectra. This result indicated the existence of a hidden electronic excited state in the vicinity of the lowest excited singlet (S1) state of the anionic form of the GFP chromophore, which is the origin of the blue shift of the two-photon absorption spectra as well as two-photon fluorescence excitation spectra.
authors.affiliation: Molecular Spectroscopy Laboratory, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Haruko
authors.initials: H
authors.lastname: Hosoi
authors.forename: Shoichi
authors.initials: S
authors.lastname: Yamaguchi
authors.forename: Hideaki
authors.initials: H
authors.lastname: Mizuno
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyawaki
authors.forename: Tahei
authors.initials: T
authors.lastname: Tahara
mesh_terms: D004583:Electrons; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D015394:Molecular Structure; D013053:Spectrophotometry; D055672:Static Electricity
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins