Enhancement of the structure and biochemical function of cyclomaltodextrinase from the Anoxybacillus flavithermus ZNU-NGA with site-directed mutagenesis.

This study was conducted to examine the role of the central domain of cyclomaltodextrinase in terms of stability, substrate specificity, becoming dodecameric form, and enzyme activity. To this end, H403R/L309V double-point mutation and T280Q single-point mutation were performed at the central domain and (ß/α)8-barrel. The results indicated that the activity of the H403R/L309V mutant at the optimal pH and temperature increased by about 25% and 40%, respectively. Plus, the irreversible thermal inactivation of the H403R/L309V mutant at 60 °C and 160 min was approximately twice of the enzyme without mutation. Both mutants underwent significant structural change relative to the wild enzyme and subsequently a significant catalytic activity. However, the catalytic efficiency (kcat/Km) of the H403R/L309V mutant increased in the presence of beta- and gamma-cyclomaltodextrin substrates compared to the wild enzyme and T280Q mutant. As a result, by applying the L309V mutant and given the smaller size of the valine, leucine spatial inhibition in the wild protein seems to decline, and also it facilitates the substrate access to active site amino acids. Moreover, as gamma substrate is larger, eliminating the effect of spatial inhibition on this substrate has a greater effect on improving the catalytic activity of this enzyme.

Site-specific mutagenesis on Mnemiopsin 2: Calcium coordination and substrate binding properties of new variants.

We used site-specific mutagenesis by targeting E179 and F190 on the structure of photoprotein Mnemiopsin 2 (Mn2) from Mnemiopsis leidyi. The tertiary structure of E179S and F190L mutants was made by the MODELLER program. Far-ultraviolet circular dichroism data showed that the overall secondary structural content of photoprotein is not changed upon mutation, however the helicity and stabilizing interactions in helical structure decreases in mutants as compared with the wild-type (WT) photoprotein. Fluorescence spectra data revealed that the tertiary structure of the mutants is more compact than that of WT Mn2. According to the heat-induced denaturation experiments data, the melting temperature (Tm ) for the unfolding of tertiary structure of the F190L variant increases by 3°C compared with that of the WT and E179S mutant. Interestingly, the conformational enthalpy of the F190L mutant (86 kcal mol-1 ) is considerably lower than those in the WT photoprotein (102 kcal mol-1 ) and E179S mutant (106 kcal mol-1 ). The significant difference in the enthalpy of the thermal unfolding process could be explained by considering that the thermally denatured state of the F190L mutant is structurally less expanded than the WT and E179S variants. Bioluminescence activity data showed that the maximum characteristic wavelengths of the mutants undergo blue shift as compared with the WT protein. Initial intensity of the F190L and E179S variants was recorded to be 137.5% and 55.9% of the WT protein, respectively.

High Oxidation Desulfurization of Fuels Catalyzed by Vanadium-Substituted Phosphomolybdate@Polyaniline@Chitosan as an Inorganic-Organic Hybrid Nanocatalyst.

From the environmental protection and human health perspectives, the design and synthesis of efficient and reusable oxidative desulfurization nanocatalysts has always been sought after by scientists and industries. In this regard, a new heterogeneous nanocatalyst (V-SPM@PANI@CH) was synthesized by immobilizing Keggin-type vanadium-substituted phosphomolybdate ([PVMo11O39]4-) (named V-SPM) clusters on the surface of polyaniline (PANI) and chitosan (CH) polymers. The features of the assembled nanocatalyst were detected by Fourier transform infrared spectroscopy, ultraviolet-visible spectroscopy, X-ray diffraction (XRD), scanning electron microscopy, and energy-dispersive X-ray spectroscopy techniques in detail. The XRD studies indicated that the average crystallite size of V-SPM@PANI@CH was estimated to be about 36 nm. The catalytic performance of V-SPM@PANI@CH was investigated in the extractive and catalytic oxidation desulfurization (ECOD) procedure of real and thiophenic model gasoline by H2O2/AcOH (volume proportion of 2:1) as an oxidizing system. The optimal desulfurization conditions for ECOD reactions were as follows: 50 mL of model/real gasoline, 0.1 g of V-SPM@PANI@CH, reaction time of 60 min, and reaction temperature of 35 °C. Under the experimental conditions outlined above and the designed ECOD system, the content of sulfur in real gasoline could decline from 0.4985 to 0.0193 wt %, which corresponds to an efficiency of 96%. Moreover, the removal percentage of aromatic hydrocarbons, including thiophene (Th), benzothiophene (BT), and di-benzothiophene (DBT) as model fuels decreases in the order of DBT ≥ BT > Th under identical operating conditions. High catalytic activity was maintained with only a slight loss during five cycles. This work offers the ECOD system (V-SPM@PANI@CH/AcOH/H2O2) for the desulfurization of liquid fuels, which had a great repercussion on the ECOD efficiency.

An in silico analysis on the photoproteins Mnemiopsin 1 and Mnemiopsin 2 to explain the experimental results.

Mnemiopsin 1 (Mn1) and Mnemiopsin 2 (Mn2) are photoproteins found in Mnemiopsis leidyi. We have tried to answer the question of whether the structural features of photoproteins can explain the observed activity data. According to the activity measurements data, they have the same characteristic wavelength. However, the initial intensity of Mn2 is significantly higher than that of Mn1, and decay time of Mn1 (0.92 s-1 ) is lower than that of Mn2 (1.46 s-1 ). The phylogenetic analysis demonstrates that, compared with Obelin and Aequorin from Obelia longissima and Aequorea victoria, respectively, a gene modification event may have caused the expansion of the N-terminal side of all photoproteins from M. leidyi. An in silico study has shown that the stability of the photoprotein-substrate complex of Mn2 is higher than that of Mn1, indicating a higher affinity of the substrate for Mn2 compared with Mn1. It was revealed that the active EF-hand loops 1 and III in Mn2 is locally more rigid compared with those in Mn1. We concluded that different stability of the photoprotein complexes leads to different initial intensity. While different patterns of the local dynamics of loops I and III may influence the decay rate.

Longer characteristic wavelength in a novel engineered photoprotein Mnemiopsin 2.

We designed two mutants of photoprotein Mnemiopsin 2 (Mn2) including M52I and V144I, where the mutations were applied in the EF-hand loops I and III. Far-UV CD measurements demonstrated that the stability of the helices in the wild-type (WT) protein is greater compared with the mutants. Heat-induced denaturation experiments in the apo-form of photoproteins showed that WT Mn2 has higher value of the enthalpy change for the unfolding process, indicating that it has more stabilizing interaction compared with mutants. According to the activity measurement data, both mutants, particularly V144I have lower initial intensity as well as slower decay rate as compared with the WT photoprotein. Importantly, it was found that V144I variant shows 25 nm of red shift in the characteristic wavelengths as compared with the WT photoprotein. This finding can be considered as an advantage for in vivo application of photoprotein for imaging purposes. It concluded that this position on loop III of Mn2 is a hotspot point for characteristic wavelength determination. However, further research on this mutant is needed for making stable variants of Mn2 with novel optical features.

Designing and construction of novel variants of Chondroitinase ABC I to reduce aggregation rate.

Chondroitinase ABC I (cABC I) can degrade inhibitory molecules for axon regrowth at the site of damage after spinal cord injury (SCI). One of the main problems in the practical application is the possibility of structural changes that lead to the inactivation of the enzyme. In current work, three variants of cABC I was designed and constructed by manipulation of a short helix conformation (Gln678-Leu679-Ser680-Gln681); where Gln residues were converted to Glu. According to the enzyme kinetics studies, the catalytic efficiency of the Q681E and double mutant (Q678E/Q681E) increases in comparison with WT enzyme; while that of Q678E decreases. It was also shown that the rate of the inactivation of the enzyme variants over time is greater in WT and Q678E variants than that of the Q681E and double mutant. Negative values of entropy change of thermal inactivation measurements; demonstrate that inactivation of the WT and Q678E variants are mainly originated from aggregation. These observations can be explained by considering the repulsive electrostatic interaction between enzyme molecules that prevents protein aggregation over time. It is concluded that increasing the solubility of the Q681E and double mutant via favorable interactions of surface-exposed charged residues with dipole momentum of water molecules accompanied by the presence of intermolecular repulsive electrostatic interaction leads to decreasing the rate of aggregation in both long-term storage and heat-induced structural changes.

Heat shock protein 70 modulates neural progenitor cells dynamics in human neuroblastoma SH-SY5Y cells exposed to high glucose content.

In the current experiment, detrimental effects of high glucose condition were investigated on human neuroblastoma cells. Human neuroblastoma cell line SH-SY5Y were exposed to 5, 40, and 70 mM glucose over a period of 72 h. Survival rate and the proliferation of cells were analyzed by MTT and BrdU incorporation assays. Apoptosis was studied by the assays of flow cytometry and PCR array. In order to investigate the trans-differentiation capacity of the cell into mature neurons, we used immunofluorescence imaging to follow NeuN protein level. The transcription level of HSP70 was shown by real-time PCR analysis. MMP-2 and -9 activities were shown by gelatin Zymography. According to data from MTT and BrdU incorporation assay, 70 mM glucose reduced cell viability and proliferation rate as compared to control (5 mM glucose) and cells treated with 40 mM glucose (P < 0.05). Cell exposure to 70 mM glucose had potential to induced apoptosis after 72 h (P < 0.05). Our results also demonstrated the sensitivity of SH-SY5Y cells to detrimental effects of high glucose condition during trans-differentiation into mature neuron-like cells. Real-time PCR analysis confirmed the expression of HSP70 in cells under high content glucose levels, demonstrating the possible cell compensatory response to an insulting condition (pcontrol vs 70 mM group <0.05). Both MMP-2 and -9 activities were reduced in cells being exposed to 70 mM glucose. High glucose condition could abrogate the dynamics of neural progenitor cells. The intracellular level of HSP70 was proportional to cell damage in high glucose condition.

Negative net charge of EF-hand loop I can affect both calcium sensitivity and substrate binding pattern in mnemiopsin 2.

Mnemiopsin 2 from Mnemiopsis leidy has three Ca2+-binding motifs and has luminescence properties in the presence of calcium and coelenterazine. It has been reported that the pattern of calcium binding among EF-hand loops of various photoproteins is different. Here, we designed and constructed two variants of mnemiopsin 2 (E50G/D47N and E50G/E53T mutants) with modified EF-hand I in which the negative charge in the first loop was reduced. According to the activity measurements, the initial intensity of mutants decreases; while the decay rate increases in E50G/D47N. We concluded that the presence of negative charge at positions 47 and 53 of mnemiopsin 2 is critical for both calcium coordination and the interaction of the substrate with the core structure of mnemiopsin 2. Structural studies accompanied by equilibrium denaturation experiments were also performed and it was found that negative charges at the aforementioned positions also have structural consequences which can affect the conformational stability of photoproteins.

A Novel Encapsulation Approach to Enhance the Delivery and Antitumor Activity of Docetaxel in Breast Cancer Therapy.

Docetaxel (DTX) is one of the most potent anticancer drugs but its extensive side effects necessitate innovative formulations. In this study, we aimed to investigate the expression pattern of apoptotic proteins, cell cycle arrest, and apoptosis induction after treatment with encapsulated DTX in alginate-chitosan nanoparticles in both breast cancer cells (MCF-7) and peripheral blood mononuclear cells (PBMCs). The characterization of the nanoparticles revealed a spherical shape with a size <50 nm, a hydrodynamic diameter of 200 nm, a Polydispersity Index of 0.5, and an encapsulation efficiency of 98.75 %. The free drug was released completely within 11 h while encapsulated DTX was released only 34 % in 96 h. The encapsulated drug indicated higher cytotoxicity on MCF-7 cells and the half inhibitory concentration (IC50) value was 2 µg/ml after 72 h. Quantitative real-time PCR demonstrated a significant increase in cell death as the expression of apoptosis regulatory protein (Bcl-2) was downregulated with no impact on Bax in the MCF-7 cells. A notable decrease in the expression pattern of pro-inflammatory cytokine (IL-1ß) in PBMCs indicated less inflammation induction. Flow cytometry analysis revealed that the newly formulated drug induced less opoptosis in PBMCs than the free DTX. Cell cycle arrest in the sub-G1 phase was observed for the free drug while the encapsulated drug exhibited no significant changes. Our results suggest the high toxicity of the formulated drug in contrast to the free DTX on the MCF-7 cell line, minimal blood cell side effects, and no inflammation positioning it as a promising alternative to free docetaxel.

Improvement of Lutein Production in Auxenochlorella protothecoides Using Its Genome-Scale Metabolic Model and a System-Oriented Approach.

Lutein is a valuable metabolite widely used in the food, pharmaceutical, cosmetic, and aquaculture industries. Marigold flowers are the most common source of commercial lutein, but cultivation area, weather conditions, and high manpower costs are among the disadvantages of lutein production from marigold flowers. Microalgae are an excellent alternative to plant sources of lutein as they do not have the limitations of plant extraction. Auxenochlorella protothecoides is a promising candidate for commercial production of lutein. In the present research, a genome-scale metabolic model was applied to introduce some strategies to improve lutein production in A. protothecoides. The effective reactions to improve lutein production were determined based on analysis of multiple optimal solutions. The enzymatic regulators of candidate reactions were identified using the BRENDA database. The effect of 13 activators was investigated experimentally. Our results showed that sodium citrate has the greatest effect on lutein production, so it was introduced as the most effective compound for increasing lutein production by A. protothecoides.

Enzyme Kinetics Features of the Representative Engineered Recombinants of Chondroitinase ABC I.

Chondroitinase ABC I (cABC I) from Proteus vulgaris is an important enzyme in medicinal biotechnology due to its ability to help axon regeneration after spinal cord injury. Its practical application involves solving several problems at the molecular and cellular levels. Structurally, most residues at the C-terminal domain of cABC I are arranged as organized strands, and only a small fraction of residues have helical conformation. The structural and functional features of modified residues on two specific helix fragments have previously been reported. The single mutant M889K has been combined with L679S and L679D mutants to make enzyme variants containing simultaneously modified helix. Here, the pH stability and temperature-based analysis of the transition state structure for the catalysis reaction were investigated. We found that double mutant L679D/M889K is the better choice to use in physiological conditions due to its higher pH stability at physiological pH as well as its different optimum temperature as compared with the (wild-type) WT protein. According to Arrhenius's analysis, the values of the Gibbs free energy of the transition state (∆G#) are not changed upon mutation. However, the relative contribution and absolute values of the enthalpy and entropy change to the total value of ∆G#, varied between the WT and mutants.

The novel anti-cancer feature of Brazzein through activating of hTLR5 by integration of biological evaluation: molecular docking and molecular dynamics simulation.

Many of plant proteins exhibit the properties similar to the antitumor proteins although the anticancer activity of Brazzein on modulating the autophagy signaling pathway has not been determined so far. The present study aimed to develop a simplified system to enable the rational design of the activating extracellular domain of human Toll-like receptor 5 (hTLR5). To identify the anticancer effect of Brazzein, HADDOCK program and molecular dynamics (MD) simulation were applied to examine the binding of the wild type (WT) and p.A19K mutant of Brazzein to the TLR5. The expression of MAP1S and TNF-α genes was estimated based on real-time PCR. The results clearly confirmed that the WT of Brazzein activated hTLR5 in the MCF-7 cell line since the genes were more and significantly less expressed in the cells treated with the WT and p.A19K mutant than the control, respectively. The snapshots of MD simulation exhibit the consistent close interactions of hTLR5 with the two helices of Brazzein on its lateral side. The results of per residue-free energy decomposition analysis substantiate those of intermolecular contact analysis perfectly one. We propose that the WT of Brazzein can act as an antitumor drug candidate.

Comparing similar versions of a connecting helix on the structure of Chondroitinase ABC I.

Regarding the existence of similar helices on the structure of different proteins, recently, novel variants of Chondroitinase ABC I (cABC I) have been constructed, where a representative helix between two structural motifs in Chondroitinase ABC I from Proteus vulgaris has been replaced by similar versions of helices found in other proteins. The previous study has revealed that the structural features and the activity of double mutants M886A/G887E (inspired by the 30 S ribosomal protein S1 from Geminocystis herdmanii) and M889I/Q891K (inspired by the chondroitin lyase from Proteus mirabilis) is comparable with that of wild-type (WT) cABC I. Here, the kinetic parameters of the enzyme activity for the WT and double mutants were determined. Of the recombinant double mutants, M889I/Q891K gave the highest catalytic efficiency with the kcat/Km value of approximately 2.3-fold increase, as compared with the WT and M886A/G887E. Modeling of experimental data showed that the mechanism of the heat-induced structural alteration, and the enzyme-substrate complex formation, changed upon mutation. These natural versions of the connecting helix can be used as an efficient linker in protein engineering studies as well as those investigations involving the use of biological linkers.

A unique EF-hand motif in mnemiopsin photoprotein from Mnemiopsis leidyi: implication for its low calcium sensitivity.

Up to now, all reported Ca(2+)-regulated photoproteins, except for mnemiopsin, have been cloned and expressed in Escherichia coli. In this study, the cDNA for an isotype of mnemiopsin, from the ctenophore Mnemiopsis leidyi, has been cloned, sequenced, and functionally expressed. The full length cDNA encoding mnemiopsin of M. leidyi was 624 bp open reading frame encoding a protein of 207 amino acid residues with calculated molecular mass of ∼24 kDa. The deduced amino acid sequence showed 90% and 84% identity to berovine (from ctenophore Beroe abyssicola) and bolinopsin 2 (from the ctenophore Bolinopsis infundibulum) respectively. In contrast to all known EF-hand in photoproteins, a unique EF-hand motif was found in mnemiopsin, in which a conserved glycine is substituted with glutamic acid. According to the results, the optimum pH was 9.0, time course of regeneration was 15 h and its Ca(2+) sensitivity was lower than aequorin. Results of pK(a) calculation for ionizable residues, motif scan and hydrophobic interactions of cavity aromatic residues of mnemiopsin in comparison with aequorin showed different patterns in these two photoproteins. In addition, experimental results are confirmed with the theoretical studies.

Bioinformatics and experimental studies on the structural roles of a surface-exposed α-helix at the C-terminal domain of Chondroitinase ABC I.

A series of single and double mutants generated on residues of a surfaced-exposed helix at the C-terminal domain of chondroitinase ABC I (cABC I) from proteus vulgaris. M886A, G887E, and their respective double mutant, MA/GE were inspired by the sequence of a similar helix segment in 30S ribosomal protein S1. Additionally, M889I, Q891K, and the corresponding double mutant, MI/QK, were made regarding the sequence of a similar helix in chondroitin lyase from Proteus mirabilis. Circular dichroism spectra in the far-UV region, demonstrate that the ordered structure of wild-type (WT), and double mutants are the same; however, the helicity of the ordered structures in MI/QK is higher than that of the WT enzyme. When compared with the single mutants, the double mutants showed higher activity, and that the activity of MI/QK is higher than that of the WT enzyme. Heat-induced denaturation experiments showed that the stability of the tertiary structure of double mutants at moderate temperatures is higher compared with the WT, and single mutants. It concluded that this helix can be considered as one of the hot spots region that can be more manipulated to obtain improved variants of cABC I.

An evolution-based designing and characterization of mutants of cyclomaltodextrinase: Molecular modeling and spectroscopic studies.

Cyclomaltodextrinase (CDase) is a member of the alpha-amylase family GH13, the subfamily GH13_20. In addition to CDase and neopullulanase, this subfamily also contains maltogenic amylase. They have common structural features, but different substrate specificity. In current work, a combination of bioinformatics and experimental tools were used for designing and constructions of single and double mutants of a new variant of CDase from Anoxybacillus flavithermus. Considering the evolutionary variable positions 123 and 127 at the dimer interface of subunits in the alpha-amylase family, these positions in CDase were modified and three mutants, including A123V, C127Q and A123V/C127Q were constructed. The tertiary structure of WT and mutants were made with the MODELLER program, and the phylogenetic tree of homologous protein sequences was built with selected programs in Phylip package. Enzyme kinetic studies revealed that the catalytic efficiency of mutants, especially double one, is lower than the WT enzyme. Heat-induced denaturation experiments were monitored by measuring the UV/Vis signal at 280 nm, and it was found that WT protein is structurally more stable at 25 °C. However, it is more susceptible to changes in temperature compared to the double mutant. It was concluded that the positions 123 and 127 at the dimeric interface of CDase, not only could affect the conformational stability; but also; the catalytic properties of the enzyme by setting up the active site configuration in the dimeric state.

Determination and evaluation of secondary structure content derived from calcium-induced conformational changes in wild-type and mutant mnemiopsin 2 by synchrotron-based Fourier-transform infrared spectroscopy.

Mnemiopsin 2 from a luminous ctenophore with two functional EF-hand motifs is a calcium-regulated photoprotein that is responsible for emitting a bright blue bioluminescence upon reacting with coelenterazine and calcium ions in Mnemiopsis leidyi. Synchrotron radiation-based Fourier-transform infrared (SR-FTIR) spectroscopy was applied to analyze the distribution of secondary structures, the conformational changes resulting from calcium binding and the structural stabilities in wild-type mnemiopsin 2, as well as its mutant type that possesses three EF-hand motifs. The distribution of secondary structures of these proteins indicates that mutant apo-mnemiopsin 2 has a more stable secondary structure than the wild-type. Analyses of the SR-FTIR spectra revealed that the conformational changes at the secondary structures of both mnemiopsin 2 depend on the calcium concentrations, such that the most noticeable changes in structures of wild-type and mutant mnemiopsin 2 occur at optimum concentrations 6 and 2 mM of calcium chloride, respectively. The addition of calcium to both proteins increases the proportions of their secondary structures in the amide I and II regions. The major amide I bands in the IR spectra of both mnemiopsin­calcium complexes shift towards smaller wavenumbers, whereas their main amide II bands are identified at larger wavenumbers.

Structural and functional consequences of replacement of His403 with Arg near the catalytic site of Anoxybacillus flavithermus cyclomaltodextrinase.

The hydrolytic activity of a thermophilic cyclomaltodextrinase (CMD) from Anoxybacillus flavithermus ZNU-NGA and a representative single mutant were investigated against soluble substrates including α-, ß- and γ-cyclomaltodestrines (CDs). Based on the occurrence of arginine (Arg) at position 403 in some homologue proteins, His403 in Wild-type (WT) CMD was replaced with Arg (H403R variant) with site-directed mutagenesis procedures. According to bioinformatics data, Arg403 in mutant protein is located near Glu357 as one of the catalytic residues in a manner that they are able to create a medium-range attractive electrostatistic interaction. Structural studies by Far UV-CD showed that this mutation is accompanied by conversion of a small fraction of α-helix to ß-form structure. Fluorescence data reveals that, the hydrophobic regions at the surface of protein, as the binding sites for ANS (8-Anilinonaphthalene-1-sulfonic acid) increase in mutant protein, demonstrating relative inflation of H403R variant compared with WT protein. However, the polarity of microenvironment around chromophores did not change upon mutation. Activity measurement in different ranges of pH and temperatures showed that the optimum values of pH and temperature in mutant enzyme is the same as WT enzyme, however; the activity at optimum points increased in H403R variant. It was also revealed that the H403R variant had slightly improved catalytic efficiency for γ-CD. The same value of activation parameters for both protein variants indicates that mutation does not alter the mechanism of catalysis during enzyme-substrate formation.

Hyaluronic acid production enhancement via genetically modification and culture medium optimization in Lactobacillus acidophilus.

Hyaluronic acid (HA) is a natural polymer with various molecular weights that specify multiple biological roles. Traditionally, HA is obtained from animal waste and conventional pathogenic streptococci. However, there are challenges in these processes such as the presence of exotoxins, hyaluronidase, and viral contamination. In order to reduce these problems, this study was conducted to produce HA using recombinant bacterium that is generally recognized as safe (GRAS), and thereafter increase production through experimental design. At first, some lactic acid bacteria were screened and evaluated for HA production. Accordingly, among the selected bacteria, Lactobacillus acidophilus PTCC1643 produced about 0.25 g HA/L in the 48th hour of cultivation, and was thus selected as an alternative host for heterologous HA production. An expression vector containing HA synthase genes was transformed into L. acidophilus by electroporation. Consequently, HA production increased to 0.4 g/L. Eventually, response surface method (RSM) was used, which increased HA production to 1.7 g/L. This is approximately 7-fold higher than that produced at first. The resulting HA was characterized by FTIR spectroscopy and its molecular weight was estimated using agarose gel electrophoresis. In conclusion, L. acidophilus could be a safe, effective, and novel HA producer with industrial potential and commercial prospects.

Molecular mechanisms governing the evolutionary conservation of Glycine in the 6th position of loops ΙΙΙ and ΙV in photoprotein mnemiopsin 2.

Photoproteins in their functional form are complexed noncovalently with 2-hydroperoxycoelenterazine. A conformational change upon coordination of Ca+2 ions with their EF-hand loops leads to oxidation of substrate and emission of light. In all photoproteins, EF-hand loops Ι, ΙΙΙ and ΙV have standard sequence for binding to Ca+2 ion, however the second one is not able for Ca+2 coordination. Sequence analysis of Mnemiopsin 2 and other known photoproteins shows that Glutamate (Glu) is occurred in the 6th position of its first EF-hand loop, but this position in other loops of mnemiopsin 2 and all functional loops of other photoproteins is occupied by Glycine (Gly). Here we designed and made single and double mutants where Gly residue at the 6th positions of loops ΙΙΙ and ΙV of mnemiopsin 2 was replaced with Glu. According to the activity measurements, wild-type (WT) and G142E variants have more initial luminescence intensity than G176E and double mutants; while WT and G176E have higher values of half decay time when compared with G142E and double mutants. According to the isothermal denaturation experiments, all protein variants are structurally more stable than WT mnemiopsin 2 and that the stabilizing effects of single mutants are paired resulting in more stability of double mutant against urea denaturation. We concluded that simultaneous occurrence of Gly in the 6th position of loops ΙΙΙ and ΙV is essential for evolutionary adjustment of initial intensity and decay rate of luminescence emission via affecting the interaction of the core structure of photoprotein with coelenteramide and binding affinity of Ca+2 to the corresponding loops, respectively.