Bioinformatics investigation of the structure and function of photoprotein mnemiopsin۲ following Glutamine ۲۳ substitutions using a site-directed mutagenesis

Ctenophores are bioluminescent marine organisms that are found all over the world. Light emission in these organisms is carried out by Ca۲+-regulated photoproteins. Ca۲+ is not essential for light emission in these photoproteins. However, The light-yielding reaction proceeds at a very slow rate in the absence of Ca۲+, and the light intensity is increased up to ۱ million-fold with the addition of Ca۲+. One of the main applications of Ca۲+-regulated photoproteins, due to their ability to emit light upon binding to calcium, is their use for detecting calcium ions in biological systems. These photoproteins consist of a single polypeptide chain and are structurally compact and globular. Mnemiopsin ۲ is a Ca۲+-regulated photoprotein extracted from Mnemiopsis leidyi. Light emotion in this photoprotein is blue light, by coelenterazine as a substrate in the presence of calcium ions. Photoprotein mnemiopsin ۲ has three active motifs; EF-hand I, III, and IV, which are located in ۴۵–۵۶, ۱۳۷–۱۴۸, and ۱۷۱–۱۸۲, respectively. The Q۲۳E mutation was investigated to increase the negative surface charge for enhanced Ca۲+ sensitivity in mnemiopsin ۲. Therefore, The three-dimensional structure of the mutant was made with the Modeller program V. ۱۰.۴, and the best structure was selected and evaluated using ModEval and SAVES. The VADAR and ProtParam servers were used to Evaluate the secondary structure of the protein, structure stability, and physicochemical properties. The Kyte & Doolittle hydropathy plot was obtained using the Prot Scale server. Then, the graphical form of the desirable model was drawn using the UCSF Chimera software, and the compactness of the protein's tertiary structure was examined. Finally, the selected mutant model was compared with the native model. The results indicate a decrease in the folding free energy of the protein with the Q۲۳E mutation, and as a result, it is more stable than the wild-type protein. Also In terms of hydrophobicity, it has not changed. It seems that by reducing the isoelectric point of the mutated protein due to the substitution of an acidic amino acid and an increase in the protein's accessible surface area, binding to Ca۲+ increases.

کلیدواژه ها:

calcium sensitivity ، Mnemiopsin ۲ ، molecular modeling ، photoprotein ، site-directed mutagenesis