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Evidence for the isomerization and decarboxylation in the photoconversion of the red fluorescent protein DsRed.
J Am Chem Soc. 2005 Jun 29; 127(25):8977-84.JA

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+Show Affiliations

Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium. satoshi_habuchi@hms.harvard.eduNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15969574

Citation

Habuchi, Satoshi, et al. "Evidence for the Isomerization and Decarboxylation in the Photoconversion of the Red Fluorescent Protein DsRed." Journal of the American Chemical Society, vol. 127, no. 25, 2005, pp. 8977-84.
Habuchi S, Cotlet M, Gensch T, et al. Evidence for the isomerization and decarboxylation in the photoconversion of the red fluorescent protein DsRed. J Am Chem Soc. 2005;127(25):8977-84.
Habuchi, S., Cotlet, M., Gensch, T., Bednarz, T., Haber-Pohlmeier, S., Rozenski, J., Dirix, G., Michiels, J., Vanderleyden, J., Heberle, J., De Schryver, F. C., & Hofkens, J. (2005). Evidence for the isomerization and decarboxylation in the photoconversion of the red fluorescent protein DsRed. Journal of the American Chemical Society, 127(25), 8977-84.
Habuchi S, et al. Evidence for the Isomerization and Decarboxylation in the Photoconversion of the Red Fluorescent Protein DsRed. J Am Chem Soc. 2005 Jun 29;127(25):8977-84. PubMed PMID: 15969574.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Evidence for the isomerization and decarboxylation in the photoconversion of the red fluorescent protein DsRed. AU - Habuchi,Satoshi, AU - Cotlet,Mircea, AU - Gensch,Thomas, AU - Bednarz,Teresa, AU - Haber-Pohlmeier,Sabina, AU - Rozenski,Jef, AU - Dirix,Gunter, AU - Michiels,Jan, AU - Vanderleyden,Jos, AU - Heberle,Joachim, AU - De Schryver,Frans C, AU - Hofkens,Johan, PY - 2005/6/23/pubmed PY - 2005/10/27/medline PY - 2005/6/23/entrez SP - 8977 EP - 84 JF - Journal of the American Chemical Society JO - J. Am. Chem. Soc. VL - 127 IS - 25 N2 - 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. SN - 0002-7863 UR - https://www.unboundmedicine.com/medline/citation/15969574/Evidence_for_the_isomerization_and_decarboxylation_in_the_photoconversion_of_the_red_fluorescent_protein_DsRed_ L2 - https://dx.doi.org/10.1021/ja047023o DB - PRIME DP - Unbound Medicine ER -