A Bilirubin-Inducible Fluorescent Protein from Eel Muscle

Abstract

The fluorescent protein toolbox has revolutionized experimental biology. Despite this advance, no fluorescent proteins have been identified from vertebrates, nor has chromogenic ligand-inducible activation or clinical utility been demonstrated. Here, we report the cloning and characterization of UnaG, a fluorescent protein from Japanese eel. UnaG belongs to the fatty-acid-binding protein (FABP) family, and expression in eel is restricted to small-diameter muscle fibers. On heterologous expression in cell lines or mouse brain, UnaG produces oxygen-independent green fluorescence. Remarkably, UnaG fluorescence is triggered by an endogenous ligand, bilirubin, a membrane-permeable heme metabolite and clinical health biomarker. The holoUnaG structure at 1.2 Å revealed a biplanar coordination of bilirubin by reversible π-conjugation, and we used this high-affinity and high-specificity interaction to establish a fluorescence-based human bilirubin assay with promising clinical utility. UnaG will be the prototype for a versatile class of ligand-activated fluorescent proteins, with applications in research, medicine, and bioengineering.

Insights into the Biosynthesis and Assembly of Cryptophycean Phycobiliproteins

“…Additionally, there are three more bilin-binding members of the calycin superfamily, whose crystal structures were recently published. UnaG originates from the Japanese eel Anguilla japonica, and its green fluorescence is based on a bound bilirubin molecule (71). Furthermore, the structure of the PBP lyase TeCpcS was determined within the National Institutes of Health Protein Structure Initiative in 2007 and published by Kronfel et al (68).…”

Structure and Mechanism of the Phycobiliprotein Lyase CpcT

“…A more extended chromophore conformation in CpcS than in CpcT would be in line with the red shift and increase of the long wavelength band in the absorption spectra of PCB bound to CpcS (61) and of phycoerythrobilin bound to CPES (29). A twisting of the two halves by 90°at the central methine bridge, as in bilirubin (57,66), however, would interrupt the conjugation and result in a strong blue shift (50).…”

“…7 and 8). Unlike biliverdins and PCB, isolated bilirubin is very hydrophobic, and strong internal H-bonds (58, 59) must be disrupted inside a chromoprotein and stabilized by hydrogen bonds to buried residues (57).…”

New Biological Frontiers Illuminated by Molecular Sensors and Actuators

“…Alternatively, fluorescent proteins can be transformed into sensors when the fluorescence is placed under the control of ligand binding. Miyawaki presented the example of UnaG, a protein found in eels that becomes fluorescent upon bilirubin binding (6). Perhaps the most famous fluorescent protein sensors are the calcium sensors, which gave rise to the discovery of calcium spikes and waves.…”