Adjustment of local conformational flexibility and accessible surface area alterations of Serine128 and Valine183 in mnemiopsin

• Bioluminescence activity in mnemiopsin is dependent on the stability of chromophore within the cavity.
• The Bioluminescence activity of S128G and V183T variants decreases relative to WT protein.
• Thermal unfolding measurements with DSC indicates that all variant unfolded via intermediate state.

We used a combination of experimental and bioinformatic studies to elucidate the importance of Serine128 and Valine183 on the activity and thermal stability of mnemiopsin 1 by substitution of S128 and V183 with glycine and threonine, respectively (S128G and V183T mutants). Luminescence emissions of S128G and V183T were reduced to 71.6% and 46.6% with respect to the original activity of the wild type protein. According to circular dichroism (CD) measurements, compactness of mutants decreased in comparison with wild type (WT) protein. Differential scanning calorimetry (DSC) indicated that Tm values of thermal unfolding are not changed significantly upon mutation. Herein, we suggest that the protein variants unfold through molecular association and intermediate states. Bioinformatic studies revealed that local fluctuation of residues in S128G increased with respect to WT protein. However, S128G mutation leads to increment of the accessible surface area of lysine188. Therefore, this change is thermodynamically favorable. Finally, both experimental and theoretical studies showed a delicate balance between all structural alterations, determining total conformational stability of the protein.

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