Background: Marine sessile organisms display a color palette that is the result of the
expression of fluorescent and non-fluorescent proteins. Fluorescent proteins have uncovered
transcriptional regulation, subcellular localization of proteins, and the fate of cells during
development. Chromoproteins have received less attention until recent years as bioreporters. Here,
we studied the properties of aeBlue, a a 25.91 kDa protein from the anemone Actinia equina.
Objective: To assess the properties of aeBlue chromoprotein under different physicochemical
Methods: In this article, during the purification of aeBlue we uncovered that it suffered a color shift
when frozen. We studied the color shift by different temperature incubation and physicochemical
conditions and light spectroscopy. To assess the possible structural changes in the protein, circular
dichroism analysis, size exclusion chromatography and native PAGE was performed.
Results: We uncover that aeBlue chromoprotein, when expressed from a synthetic construct in
Escherichia coli, showed a temperature dependent color shift. Protein purified at 4 °C by metal
affinity chromatography exhibited a pinkish color and shifts back at higher temperatures to its
intense blue color. Circular dichroism analysis revealed that the structure in the pink form of the
protein has reduced secondary structure at 4 °C, but at 35 °C and higher, the structure shifts to a
native conformation and Far UV- vis CD spectra revealed the shift in an aromatic residue of the
chromophore. Also, the chromophore retains its properties in a wide range of conditions (pH,
denaturants, reducing and oxidants agents). Quaternary structure is also maintained as a tetrameric
conformation as shown by native gel and size exclusion chromatography.
Conclusion: Our results suggest that the chromophore position in aeBlue is shifted from its native
position rendering the pink color and the process to return it to its native blue conformation is