title: Evidence for the isomerization and decarboxylation in the photoconversion of the red fluorescent protein DsRed.
abstract: Recently, it has been shown that the red fluorescent protein DsRed undergoes photoconversion on intense irradiation, but the mechanism of the conversion has not yet been elucidated. Upon irradiation with a nanosecond-pulsed laser at 532 nm, the chromophore of DsRed absorbing at 559 nm and emitting at 583 nm (R form) converts into a super red (SR) form absorbing at 574 nm and emitting at 595 nm. This conversion leads to a significant change in the fluorescence quantum yield from 0.7 to 0.01. Here we demonstrate that the photoconversion is the result of structural changes of the chromophore and one amino acid. Absorption, fluorescence, and vibrational spectroscopy as well as mass spectrometry suggest that a cis-to-trans isomerization of the chromophore and decarboxylation of a glutamate (E215) take place upon irradiation to form SR. At the same time, another photoproduct (B) with an absorption maximum at 386 nm appears upon irradiation. This species is assigned as a protonated form of the DsRed chromophore. It might be a mixture of several protonated DsRed forms as there is at least two ways of formation. Furthermore, the photoconversion of DsRed is proven to occur through a consecutive two-photon absorption process. Our results demonstrate the importance of the chromophore conformation in the ground state on the brightness of the protein as well as the importance of the photon flux to control/avoid the photoconversion process.
authors.affiliation: Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium. satoshi_habuchi@hms.harvard.edu
authors.forename: Satoshi
authors.initials: S
authors.lastname: Habuchi
authors.forename: Mircea
authors.initials: M
authors.lastname: Cotlet
authors.forename: Thomas
authors.initials: T
authors.lastname: Gensch
authors.forename: Teresa
authors.initials: T
authors.lastname: Bednarz
authors.forename: Sabina
authors.initials: S
authors.lastname: Haber-Pohlmeier
authors.forename: Jef
authors.initials: J
authors.lastname: Rozenski
authors.forename: Gunter
authors.initials: G
authors.lastname: Dirix
authors.forename: Jan
authors.initials: J
authors.lastname: Michiels
authors.forename: Jos
authors.initials: J
authors.lastname: Vanderleyden
authors.forename: Joachim
authors.initials: J
authors.lastname: Heberle
authors.forename: Frans C
authors.initials: FC
authors.lastname: De Schryver
authors.forename: Johan
authors.initials: J
authors.lastname: Hofkens
mesh_terms: D003653:Decarboxylation; D007536:Isomerism; D007834:Lasers; D008164:Luminescent Proteins / Q000737:chemistry* / Q000528:radiation effects*; D015394:Molecular Structure; D010782:Photolysis; D013997:Time Factors
chemical_list: D008164:Luminescent Proteins; C413663:fluorescent protein 583