A Dual-Ligand-Modulable Functional Protein Based on Ligand-inducible Green Fluorescent Protein and Calmodulin

Abstract

UnaG, a superfamily of fatty-acid-binding protein (FABP), is a green-emitting fluorescent protein that utilizes unconjugated bilirubin (BR) as its fluorophore. Physiologically, BR is a notable antioxidant to scavenge reactive oxygen species in biological membranes, although excessive accumulation of BR causes several clinical symptoms such as jaundice. The optimal regulation of the BR concentration in biological tissues would result in clinical therapies for aging as well as reduce risks of clinical symptoms. However, UnaG hardly releases BR because of its high affinity for BR (Kd= 98 pM). In this study, we generated Ca2+-inducible BR releasing protein by genetically insertion of calmodulin (CaM) into UnaG. We speculated that the insertion of calmodulin (CaM) near the BR binding pocket of UnaG would result in the production of a novel fluorescent protein that releases the fluorophore in a Ca2+-dependent manner. In addition, as the site is close to Ser80/Asp81 residues, which anchor the propionate carboxylate from the exo-vinyl dipyrrinone of BR, the Ca2+-dependent conformational change on the inserted CaM was expected to affect the BR affinity. The resultant UnaG/CaM hybrid protein, named BReleaCa (BR+releaser+Ca2+), is a dual-ligand modulable fluorescent protein; binding of the fluorogenic ligand BR is negatively regulated by the other ligand, Ca2+ion. The affinity for BR differed by three orders of magnitude between the Ca2+-free state (Kd= 9.70 nM) and Ca2+-saturated state (Kd= 9.65 μM). The chimeric protein can release nano- to micro-molar levels of BR with Ca2+control. Such a protein hybridization technique will be applicable as a method of changing the ligand binding properties of different ligand-inducible protein.