Investigating the effect of electric dipole of antiparallel alpha helices on the structure and function of photoprotein Mnemiopsin 2

Norouzi, Fatemeh; Shirdel, S. Akram; Norouzi, Zahra; Jafarian*, Vahab; Khalifeh, Khosrow

Investigating the effect of electric dipole of antiparallel alpha helices on the structure and function of photoprotein Mnemiopsin 2

Authors

Fatemeh Norouzi ¹

S. Akram shirdel ¹

Zahra Norouzi ¹

Vahab Jafarian* ²

Khosrow Khalifeh ¹

¹ University of Zanjan

² University of Guilan

Abstract

In this study, the electrostatic interactions between the third and fourth antiparallel helices of Mnemiopsin 2 have studied. Due to the distribution of the positive and negatively charged residues at the C- and N-terminals of helices, we treated them as two antiparallel nanowires with electric dipole character. By mutating Phe87 to Arg, we aimed to enhance the electric dipole of the fourth helix. The MODELLER program was employed to generate the tertiary structure of the wild-type and mutant proteins. The expression system was prepared using the site-directed mutagenesis procedure. Activity measurements revealed that while the wild-type protein exhibited faster reaction initiation and a higher decay rate. However, the mutant displayed a significantly higher photon yield, approximately double that of the wild-type. Intrinsic fluorescence measurements indicated that the microenvironment around the chromophores in the mutant photoprotein had altered, leading to increased distance of chromophores from internal quenchers. However, the overall structure of the mutant protein became more compact, as confirmed by the ANS-based fluorescence. Heat-induced denaturation experiments showed that while the melting temperature (T_m) remained unaffected, the enthalpy change of denaturation increased significantly in the mutant, suggesting enhanced cooperativity in the intramolecular stabilizing interactions. Finally, it was concluded that increasing the cooperativity between the stabilizing interactions in the mutant protein, stabilize the native structure, leading to a higher population of functional complexes and, consequently, a higher photon yield.

Keywords

Mnemiopsin 2

photoprotein

Mutant

Fluorescence

Heat-induced unfolding

Thermodynamics

Subjects

Molecular biotechnology

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