Chromophore Formation in DsRed Occurs by a Branched Pathway

Like GFP, the fluorescent protein DsRed has a chromophore that forms autocatalytically within the folded protein, but the mechanism of DsRed chromophore formation has been unclear. It was proposed that an initial oxidation generates a green chromophore, and that a final oxidation yields the red chromophore. However, this model does not adequately explain why a mature DsRed sample contains a mixture of green and red chromophores. We present evidence that the maturation pathway for DsRed branches upstream of chromophore formation. After an initial oxidation step, a final oxidation to form the acylimine of the red chromophore is in kinetic competition with a dehydration to form the green chromophore. This scheme explains why green and red chromophores are alternative end points of the maturation pathway.