Structural Characterization of Acylimine-Containing Blue and Red Chromophores in mTagBFP and TagRFP Fluorescent Proteins Original Research Article

We determined the 2.2 Å crystal structures of the red fluorescent protein TagRFP and its derivative, the blue fluorescent protein mTagBFP. The crystallographic analysis is consistent with a model in which TagRFP has the trans coplanar anionic chromophore with the conjugated π-electron system, similar to that of DsRed-like chromophores. Refined conformation of mTagBFP suggests the presence of an N-acylimine functionality in its chromophore and single Cα-Cβ bond in the Tyr64 side chain. Mass spectrum of mTagBFP chromophore-bearing peptide indicates a loss of 20 Da upon maturation, whereas tandem mass spectrometry reveals that the Cα-N bond in Leu63 is oxidized. These data indicate that mTagBFP has a new type of the chromophore, N-[(5-hydroxy-1H-imidazole-2-yl)methylidene]acetamide. We propose a chemical mechanism in which the DsRed-like chromophore is formed via the mTagBFP-like blue intermediate.