Multi-domain GFP-like proteins from two species of marine hydrozoans.

Proteins homologous to Green Fluorescent Protein (GFP) are widely used as genetically encoded fluorescent labels. Many developments of this technology were spurred by discoveries of novel types of GFP-like proteins (FPs) in nature. Here we report two proteins displaying primary structures never before encountered in natural FPs: they consist of multiple GFP-like domains repeated within the same polypeptide chain. A two-domain green FP (abeGFP) and a four-domain orange-fluorescent FP (Ember) were isolated from the siphonophore Abylopsis eschscholtzii and an unidentified juvenile jellyfish (order Anthoathecata), respectively. Only the most evolutionary ancient domain of Ember is able to synthesize an orange-emitting chromophore (emission at 571 nm), while the other three are purely green (emission at 520 nm) and putatively serve to maintain the stability and solubility of the multidomain protein. When expressed individually, two of the green Ember domains form dimers and the third one exists as a monomer. The low propensity for oligomerization of these domains would simplify their adoption as in vivo labels. Our results reveal a previously unrecognized direction in which natural FPs have diversified, suggesting new avenues to look for FPs with novel and potentially useful features.