The structure of the chromophore within DsRed, a red fluorescent protein from coral.
DsRed, a brilliantly red fluorescent protein, was recently cloned from Discosoma coral by homology to the green fluorescent protein (GFP) from the jellyfish Aequorea. 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.
Gross
L A
LA
Department of Pharmacology, Howard Hughes Medical Institute, University of California, San Diego, La Jolla, 92093, USA.
Baird
G S
GS
Hoffman
R C
RC
Baldridge
K K
KK
Tsien
R Y
RY
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002863:Chromogenic Compounds / Q000737:chemistry*; D003063:Cnidaria / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D008164:Luminescent Proteins / Q000737:chemistry*; D013058:Mass Spectrometry; D011487:Protein Conformation; D011489:Protein Denaturation; D000097573:Red Fluorescent Protein
D002863:Chromogenic Compounds; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein