Probing the structural determinants of yellow fluorescence of a protein from Phialidium sp.

Fluorescent proteins homologous to green fluorescent protein (avGFP) display pronounced spectral variability due to different chromophore structures and variable chromophore interactions with the surrounding amino acids. To gain insight into the structural basis for yellow emission, the 3D structure of phiYFP (λem = 537 nm), a protein from the sea medusa Phialidium sp., was built by a combined homology modeling – mass spectrometry approach. Mass spectrometry of the isolated chromophore-bearing peptide reveals that the chromophore of phiYFP is chemically identical to that of avGFP (λem = 508 nm). The experimentally acquired chromophore structure was combined with the homology-based model of phiYFP, and the proposed 3D structure was used as a starting point for identification of the structural features responsible for yellow fluorescence. Mutagenesis of residues in the local chromophore environment of phiYFP suggests that multiple factors cooperate to establish the longest-wavelength emission maximum among fluorescent proteins with an unmodified GFP-like chromophore.

► Multiple factors cooperate to establish the red-shifted spectra of phiYFP.
► Tyr203 participates in the π-π stacking interactions with the chromophore.
► The lack of hydroxyl-containing aliphatic amino acids at positions 205 and 203.
► Glu222 hydrogen bonds to the imidazolinone nitrogen via the Thr65 side chain oxygen.