Polarity change of a representative helix in coelenterazin-binding cavity of mnemiopsin 2: Functional and structural consequences

Abstract

Abstract To evaluate the effect of a positively charged residue at the end of the fourth helix on the function and stability of the folded state of mnemiopsin 2, Lys89 was replaced with Ala (K89A mutant). Its structural and functional features were investigated and compared with Wild type (WT) protein using a combination of theoretical and experimental procedures. The tertiary structures of WT and mutant photoproteins were made with MODELLER program, and were used for designing of mutation as well as explaining the results of experimental measurements. Activity measurements showed that the initial intensity of light emission is reduced, but decay time is not changed upon mutation. K89A mutant exhibited more sensitivity to calcium ion than WT protein did. As compared to the pH 9 of WT, the optimum pH for the mutant increased to 9.25. According to the circular dichroism measurements at far UV region, the total amount of helical structure is not changed upon mutation; however, the helicity of chromophores is reduced in K89A mutant, demonstrating that the secondary structural elements in WT protein are more rigid. Heat-induced denaturation data showed that the stability of the WT is greater than mutant photoprotein by 2 kcal/mol. It appears that the higher value of the dipole momentum at the axis of fourth helix in WT photoprotein leads to strengthening of intra-molecular “head-to-tail” versus “side-by-side” anti-parallel interaction between the third and fourth helices. It was concluded that dipole-dipole interaction between the helices are important factors in packaging of the substrate in the core structure of the photoprotein.