title: Mechanism Behind the Apparent Large Stokes Shift in LSSmOrange Investigated by Time-Resolved Spectroscopy.
abstract: LSSmOrange is a fluorescent protein with a large energy gap between the absorption and emission bands (5275 cm(-1)). The electronic structure of the LSSmOrange chromophore, 2-[(5-)-2-hydroxy-dihydrooxazole]-4-(p-hydroxybenzylidene)-5-imidazolinone, is affected by deprotonation of the p-hydroxybenzylidene group. We investigated LSSmOrange by time-resolved spectroscopy in the femtosecond and nanosecond range. The ground state chromophore was almost exclusively in the neutral form, which had a main absorption band at 437 nm with a small shoulder at 475 nm. The absorption at a wavelength within the former band promoted the protein to the excited state where excited state proton transfer (ESPT) could lead to deprotonation in 0.8 ps. Following ESPT, the chromophore emitted fluorescence with a maximum at 573 nm and a decay time of 3500 ps. Although deprotonation by ESPT occurs, we unexpectedly found a slow accumulation of the anionic form in the ground state upon repeated high intensity excitation. This accumulation of the anionic form was accompanied by a shift of the absorption band to 553 nm without changing the emission band. MALDI-MS revealed that this shift is accompanied by decarboxylation of E222, which is interacting with the imidazolinone ring of the chromophore. We concluded that the photoinduced decarboxylation induced a conformational change that affected local environment around the hydroxyl group, resulting in a stable deprotonated form of the chromophore.
authors.affiliation: Molecular Imaging and Photonics, Department of Chemistry, KU Leuven , Celestijnenlaan 200F, 3001 Heverlee, Belgium.
authors.forename: Eduard
authors.initials: E
authors.lastname: Fron
authors.affiliation: Laboratory of Biomolecular Network Dynamics, Biochemistry, Molecular and Structural Biology Section, Department of Chemistry, KU Leuven , Celestijnenlaan 200G, bus 2403, 3001 Heverlee, Belgium.
authors.forename: Herlinde
authors.initials: H
authors.lastname: De Keersmaecker
authors.affiliation: Molecular Imaging and Photonics, Department of Chemistry, KU Leuven , Celestijnenlaan 200F, 3001 Heverlee, Belgium.
authors.forename: Susana
authors.initials: S
authors.lastname: Rocha
authors.affiliation: Molecular Imaging and Photonics, Department of Chemistry, KU Leuven , Celestijnenlaan 200F, 3001 Heverlee, Belgium.
authors.forename: Yannick
authors.initials: Y
authors.lastname: Baeten
authors.affiliation: Molecular Imaging and Photonics, Department of Chemistry, KU Leuven , Celestijnenlaan 200F, 3001 Heverlee, Belgium.
authors.forename: Gang
authors.initials: G
authors.lastname: Lu
authors.affiliation: Molecular Imaging and Photonics, Department of Chemistry, KU Leuven , Celestijnenlaan 200F, 3001 Heverlee, Belgium.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Uji-i
authors.affiliation: Molecular Imaging and Photonics, Department of Chemistry, KU Leuven , Celestijnenlaan 200F, 3001 Heverlee, Belgium.
authors.forename: Mark
authors.initials: M
authors.lastname: Van der Auweraer
authors.affiliation: Molecular Imaging and Photonics, Department of Chemistry, KU Leuven , Celestijnenlaan 200F, 3001 Heverlee, Belgium.
authors.forename: Johan
authors.initials: J
authors.lastname: Hofkens
authors.affiliation: Laboratory of Biomolecular Network Dynamics, Biochemistry, Molecular and Structural Biology Section, Department of Chemistry, KU Leuven , Celestijnenlaan 200G, bus 2403, 3001 Heverlee, Belgium.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Mizuno
mesh_terms: D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D013050:Spectrometry, Fluorescence / Q000379:methods*