Site‐specific mutagenesis on Mnemiopsin 2: Calcium coordination and substrate binding properties of new variants

We used site‐specific mutagenesis by targeting E179 and F190 on the structure of photoprotein Mnemiopsin 2 (Mn2) from Mnemiopsis leidyi. The tertiary structure of E179S and F190L mutants was made by the MODELLER program. Far‐ultraviolet circular dichroism data showed that the overall secondary structural content of photoprotein is not changed upon mutation, however the helicity and stabilizing interactions in helical structure decreases in mutants as compared with the wild‐type (WT) photoprotein. Fluorescence spectra data revealed that the tertiary structure of the mutants is more compact than that of WT Mn2. According to the heat‐induced denaturation experiments data, the melting temperature (Tm) for the unfolding of tertiary structure of the F190L variant increases by 3°C compared with that of the WT and E179S mutant. Interestingly, the conformational enthalpy of the F190L mutant (86 kcal mol−1) is considerably lower than those in the WT photoprotein (102 kcal mol−1) and E179S mutant (106 kcal mol−1). The significant difference in the enthalpy of the thermal unfolding process could be explained by considering that the thermally denatured state of the F190L mutant is structurally less expanded than the WT and E179S variants. Bioluminescence activity data showed that the maximum characteristic wavelengths of the mutants undergo blue shift as compared with the WT protein. Initial intensity of the F190L and E179S variants was recorded to be 137.5% and 55.9% of the WT protein, respectively. Two mutants of Mnemiopsin2 including E179S and F190L were designed and constructed. Mutations affect the calcium coordination by the loop IV, leading to changing the decay rate of the luminescence activity. Due to the changing of the pattern of the intermolecular interactions, the stability of photoprotein–substrate complexes of mutants are changed leading to changes in the initial intensity of the luminescence activity.