Structural and functional effects of helix dipole moment in photoprotein Mnemiopsin 2

Here, we designed and constructed a double mutant of photoprotein Mnemiopsin 2 by targeting two antiparallel helix fragments within the structure of photoprotein. In designing of the mutation, two helices were treated as electric dipoles, and their dipole character was manipulated by substituting neutral residues with charged ones at the helix termini. Activity measurements have revealed that mutant protein exhibits a higher initial luminescence intensity compared to the wild-type photoprotein. Based on the intrinsic and extrinsic fluorescence measurements, the overall tertiary structure of mutant photoprotein becomes more compact than the wild-type photoprotein. Heat-induced unfolding experiments demonstrated that the mutant photoprotein exhibited a 3^oC higher T_m compared to the wild-type. Additionally, the enthalpy change associated with the unfolding process increases approximately two-fold, from 35 to 72 kcal/mole. Higher enthalpy change demonstrates that the stabilizing interactions in the mutant photoprotein are more cooperative and, it is less likely to undergo partial denaturation toward misfolded states at moderate temperatures. According to this model, in the same concentration of both photoprotein, more population of the mutant photoprotein are in the folded state as compared with that in the wild-type one.