TY  - JOUR
AU  - Habuchi, S.
AU  - Cotlet, M.
AU  - Gensch, T.
AU  - Bednarz, T.
AU  - Haber-Pohlmeier, S.
AU  - Rozenski, J.
AU  - Dirix, G.
AU  - Michiels, J.
AU  - Vanderleyden, J.
AU  - Heberle, J.
AU  - de Schryver, F. C.
AU  - Hofkens, J.
TI  - Evidence for the isomerization and decarboxylation in the photoconversion of the red fluorescent protein DsRed
JO  - Journal of the American Chemical Society
VL  - 127
SN  - 0002-7863
CY  - Washington, DC
PB  - American Chemical Society
M1  - PreJuSER-46162
SP  - 8977 - 8984
PY  - 2005
N1  - Record converted from VDB: 12.11.2012
AB  - 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.
KW  - Decarboxylation
KW  - Isomerism
KW  - Lasers
KW  - Luminescent Proteins: chemistry
KW  - Luminescent Proteins: radiation effects
KW  - Molecular Structure
KW  - Photolysis
KW  - Time Factors
KW  - Luminescent Proteins (NLM Chemicals)
KW  - fluorescent protein 583 (NLM Chemicals)
KW  - J (WoSType)
LB  - PUB:(DE-HGF)16
C6  - pmid:15969574
UR  - <Go to ISI:>//WOS:000230010600037
DO  - DOI:10.1021/ja047023o
UR  - https://juser.fz-juelich.de/record/46162
ER  -