The structure of the chromophore within DsRed, a red fluorescent protein from coral
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- Larry A. Gross
- Department of Pharmacology, Medical Scientist Training Program and Biomedical Sciences Program, Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, and San Diego Supercomputer Center, University of California, San Diego, La Jolla, CA 92093
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- Geoffrey S. Baird
- Department of Pharmacology, Medical Scientist Training Program and Biomedical Sciences Program, Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, and San Diego Supercomputer Center, University of California, San Diego, La Jolla, CA 92093
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- Ross C. Hoffman
- Department of Pharmacology, Medical Scientist Training Program and Biomedical Sciences Program, Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, and San Diego Supercomputer Center, University of California, San Diego, La Jolla, CA 92093
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- Kim K. Baldridge
- Department of Pharmacology, Medical Scientist Training Program and Biomedical Sciences Program, Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, and San Diego Supercomputer Center, University of California, San Diego, La Jolla, CA 92093
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- Roger Y. Tsien
- Department of Pharmacology, Medical Scientist Training Program and Biomedical Sciences Program, Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, and San Diego Supercomputer Center, University of California, San Diego, La Jolla, CA 92093
説明
<jats:p>DsRed, a brilliantly red fluorescent protein, was recently cloned from<jats:italic>Discosoma</jats:italic>coral by homology to the green fluorescent protein (GFP) from the jellyfish<jats:italic>Aequorea</jats:italic>. A core question in the biochemistry of DsRed is the mechanism by which the GFP-like 475-nm excitation and 500-nm emission maxima of immature DsRed are red-shifted to the 558-nm excitation and 583-nm emission maxima of mature DsRed. After digestion of mature DsRed with lysyl endopeptidase, high-resolution mass spectra of the purified chromophore-bearing peptide reveal that some of the molecules have lost 2 Da relative to the peptide analogously prepared from a mutant, K83R, that stays green. Tandem mass spectrometry indicates that the bond between the alpha-carbon and nitrogen of Gln-66 has been dehydrogenated in DsRed, extending the GFP chromophore by forming —C⩵N—C⩵O at the 2-position of the imidazolidinone. This acylimine substituent quantitatively accounts for the red shift according to quantum mechanical calculations. Reversible hydration of the C⩵N bond in the acylimine would explain why denaturation shifts mature DsRed back to a GFP-like absorbance. The C⩵N bond hydrolyses upon boiling, explaining why DsRed shows two fragment bands on SDS/PAGE. This assay suggests that conversion from green to red chromophores remains incomplete even after prolonged aging.</jats:p>
収録刊行物
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- Proceedings of the National Academy of Sciences
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Proceedings of the National Academy of Sciences 97 (22), 11990-11995, 2000-10-24
Proceedings of the National Academy of Sciences