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 βCβ©΅NβCβ©΅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 Cβ©΅N bond in the
acylimine would explain why denaturation shifts mature DsRed back to a
GFP-like absorbance. The Cβ©΅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.