Site-directed mutagenesis of photoprotein mnemiopsin: implication of some conserved residues in bioluminescence properties

Abstract

Mnemiopsin is a Ca(2+)-binding photoprotein from Mnemiopsis leidyi that emits a flash of blue light upon reacting with coelenterazine and Ca(2+). The light emission is a result of an intramolecular oxidation reaction. Similar to the other Ca(2+)-binding photoproteins, mnemiopsin is composed of apophotoprotein (206 amino acid residues), the imidazopyrazine chromophore, coelenterazine, and molecular oxygen. The biochemical properties of this photoprotein have been recently characterized but so far there has been no individual study on the role of critical residues. In this study, we introduced some mutations in the mnemiopsin structure for investigation of the roles of some critical residues in the substrate binding cavity, and neighboring residues in the mechanism of the reaction and the bioluminescence properties of the photoprotein. Mutants of mnemiopsin were produced by substitution of residues M77, W101 and M151. Three mutants (W101F, W101Y and M151Y mutants) had significantly reduced luminescence activity and altered bioluminescent properties (such as decay rate, Ca(2+) sensitivity, etc.), whereas the fourth (M77H mutant) lost its luminescence activity completely. Our experimental and theoretical studies suggest that residue M77 probably has structural importance and participates in stabilization of active site residues, whereas residue M151 is one of the critical mechanistic residues in ctenophore photoproteins.

QM/MM simulations provide insight into the mechanism of bioluminescence triggering in ctenophore photoproteins

“…A number of mutagenesis studies have been carried out on berovin and mnemiopsin in order to determine the key amino acid residues that surround the coelenterazine [ 28 , 51 – 53 ]. However, these studies are incomplete based on the computational results described above so further mutations were performed here in order to obtain a complete picture.…”

Improving the luminescence properties of aequorin by conjugating to CdSe/ZnS quantum dot nanoparticles: Red shift and slowing decay rate

“…Tricoir et al [62] found that by creating mutations in aequorin which increases the decay time, the tendency to calcium binding will decrease. On the other hand, Mahdavi et al [63] showed that mutations, which cause the decrease in structural rigidity of mnemiopsin, another Ca 2+ -regulated photoprotein, may result in an increase in the decay time. They suggested that transduction of conformational changes happen slowly following calcium binding, thereby the rate of the bioluminescence reaction decreases and in contrast, the decay time increases.…”

“…Various studies have shown that the change in the decay time of photoprotein can be due to the change in photoprotein's flexibility, structural rigidity and the change in calcium sensitivity [62,63]. On the other hand, it has been established that the binding of enzymes (proteins) to a support introduces the additional structural rigidity and immobilized enzymes show lower conformational flexibility, which in turn decreases their catalytic potential [64][65][66].…”

Fluorescent Protein–photoprotein Fusions and Their Applications in Calcium Imaging

“…It is commonly observed that the decay rate increases with Ca 2+ concentration. Some reports hint that the change in such emission decay is related to the structural rigidity of the photoprotein . In fact, the conjugation of quantum dots with aequorin increased its conformational rigidity, resulting in longer emission decay .…”