Title: Evidence for the isomerization and decarboxylation in the photoconversion of the red fluorescent protein DsRed
Authors: Habuchi, Satoshi ×
Cotlet, Mircea
Gensch, Thomas
Bednarz, Teresa
Haber-Pohlmeier, Sabina
Rozenski, Jef
Dirix, Gunter
Michiels, Jan
Vanderleyden, Jozef
Heberle, Joachim
De Schryver, Frans
Hofkens, Johan #
Issue Date: Jun-2005
Series Title: Journal of the American Chemical Society vol:127 issue:25 pages:8977-8984
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.
ISSN: 0002-7863
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Centre of Microbial and Plant Genetics
Medicinal Chemistry (Rega Institute)
Molecular Imaging and Photonics
× corresponding author
# (joint) last author

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