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.

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.

Effects of representative point mutations on dynamic behavior of the DISC1-Ndel1 complex: a molecular dynamics study.

It has been found that the development of schizophrenia and some other psychiatric disorders is related to defects in the normal functioning of Disrupted-In-Schizophrenia 1 (DISC1). It is a large-sized protein containing 855 residues and acts as an active hub at the core of many interactions with various proteins. On the other hand, NudE Neurodevelopment Protein 1 Like 1 (Ndel1) plays a role in nervous system development via interaction with the DISC1. It was shown that some point mutations on DISC1 have clinical implications. In line with these reports, here we have used the NMR structure of the wild-type (WT) C-terminal tail of DISC1 in complex with the N-terminal fragment of Ndel1, and have constructed the three-dimensional structures of L62Q and L29Q mutants, as the pathologic variants of the complex. The time-dependent interaction of DISC1 with Ndel1 in the WT complex and mutants was simulated by performing molecular dynamics (MD) simulation using programs in the GROMACS package. It was found that the flexibility of residues in some regions of the protein chains increases, and secondary structural changes from ordered toward unordered one leads to destabilizing of the complex in mutants. Destabilization of the complex upon substitution of Leu by Gln was also confirmed by analysis of the contact map plot.Communicated by Ramaswamy H. Sarma.

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.

Evolutionary adaptation of DHFR via expression of enzyme isoforms with various binding properties and dynamics behavior: a bioinformatics and computational study.

We compared the binding properties and dynamics of three experimentally reviewed isoforms of human dihydrofolate reductase (DHFR). The cytoplasmic variants including isoforms1 and 2 (iso1 and iso2) are produced by alternative splicing; while the mitochondrial form is located in the mitochondria. The iso1 as the canonical sequence contains 187 residues, and iso2 differs from the iso1, where it has 1-52 residues missing at the N-terminus of canonical sequence. Here, the structural models of the iso2 and mitochondrial forms were constructed by the MODELLER program using the crystal structure of the iso1 as the template. Bioinformatics analysis on ligand-bearing structures demonstrates that mitochondrial variant forms more stable complex with ligands compared with iso1 and 2, indicating their different binding properties. The root mean square fluctuation (RMSF) data suggest that C-terminus of iso1 contains two representative highly flexible fragments, while iso2 contains a highly flexible fragment at N-terminus end. Interestingly, both ends of mitochondrial variant have a degree of rigidity. Finally, the observation of differences in structural dynamics and binding properties predicts that the simultaneous existence of enzyme isoforms is a way to increase the speed of the enzyme maneuver in response to various environmental conditions. This prediction needs to be tested experimentally.

Thermostability of Ctenophore and Coelenterate Ca2+-Regulated Apo-photoproteins: A Comparative Study.

The stabilities of Ca2+-regulated ctenophore and coelenterate apo-photoproteins, apo-mnemiopsin (apo-Mne) and apo-aequorin (apo-Aeq), respectively, were compared biochemically, biophysically, and structurally. Despite high degrees of structural and functional conservation, drastic variations in stability and structural dynamics were found between the two proteins. Irreversible thermoinactivation experiments were performed upon incubation of apo-photoproteins at representative temperatures. The inactivation rate constants (kinact) at 50 °C were determined to be 0.001 and 0.004 min-1 for apo-Mne and apo-Aeq, respectively. Detailed analysis of the inactivation process suggests that the higher thermostability of apo-Mne is due to the higher activation energy (Ea) and subsequently higher values of ΔH* and ΔG* at a given temperature. According to molecular dynamics simulation studies, the higher hydrogen bond, electrostatic, and van der Waals energies in apo-Mne can validate the relationship between the thermal adaptation of apo-Mne and the energy barrier for the inactivation process. Our results show that favorable residues for protein thermostability such as hydrophobic, charged, and adopted α-helical structure residues are more frequent in the apo-Mne structure. Although the effect of acrylamide on fluorescence quenching suggests that the local flexibility in regions around Trp and Tyr residues of apo-Aeq is higher than that of apo-Mne, which results in it having a better ability to penetrate acrylamide molecules, the root-mean-square fluctuation of helix A in apo-Mne is higher than that in apo-Aeq. It seems that the greater flexibility of apo-Mne in these regions may be considered as a determining factor, affecting the thermal stability of apo-Mne through a balance between structural rigidity and flexibility.

A chaotic viewpoint-based approach to solve haplotype assembly using hypergraph model.

Decreasing the cost of high-throughput DNA sequencing technologies, provides a huge amount of data that enables researchers to determine haplotypes for diploid and polyploid organisms. Although various methods have been developed to reconstruct haplotypes in diploid form, their accuracy is still a challenging task. Also, most of the current methods cannot be applied to polyploid form. In this paper, an iterative method is proposed, which employs hypergraph to reconstruct haplotype. The proposed method by utilizing chaotic viewpoint can enhance the obtained haplotypes. For this purpose, a haplotype set was randomly generated as an initial estimate, and its consistency with the input fragments was described by constructing a weighted hypergraph. Partitioning the hypergraph specifies those positions in the haplotype set that need to be corrected. This procedure is repeated until no further improvement could be achieved. Each element of the finalized haplotype set is mapped to a line by chaos game representation, and a coordinate series is defined based on the position of mapped points. Then, some positions with low qualities can be assessed by applying a local projection. Experimental results on both simulated and real datasets demonstrate that this method outperforms most other approaches, and is promising to perform the haplotype assembly.

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.

RCOVID19: Recurrence-based SARS-CoV-2 features using chaos game representation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. It was first detected in China and was rapidly spread to other countries. Several thousands of whole genome sequences of SARS-CoV-2 have been reported and it is important to compare them and identify distinctive evolutionary/mutant markers. Utilizing chaos game representation (CGR) as well as recurrence quantification analysis (RQA) as a powerful nonlinear analysis technique, we proposed an effective process to extract several valuable features from genomic sequences of SARS-CoV-2. The represented features enable us to compare genomic sequences with different lengths. The provided dataset involves totally 18 RQA-based features for 4496 instances of SARS-CoV-2.

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.

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.

Application of Chaotic Laws to Improve Haplotype Assembly Using Chaos Game Representation.

Sequence data are deposited in the form of unphased genotypes and it is not possible to directly identify the location of a particular allele on a specific parental chromosome or haplotype. This study employed nonlinear time series modeling approaches to analyze the haplotype sequences obtained from the NGS sequencing method. To evaluate the chaotic behavior of haplotypes, we analyzed their whole sequences, as well as several subsequences from distinct haplotypes, in terms of the SNP distribution on their chromosomes. This analysis utilized chaos game representation (CGR) followed by the application of two different scaling methods. It was found that chaotic behavior clearly exists in most haplotype subsequences. For testing the applicability of the proposed model, the present research determined the alleles in gap positions and positions with low coverage by using chromosome subsequences in which 10% of each subsequence's alleles are replaced by gaps. After conversion of the subsequences' CGR into the coordinate series, a Local Projection (LP) method predicted the measure of ambiguous positions in the coordinate series. It was discovered that the average reconstruction rate for all input data is more than 97%, demonstrating that applying this knowledge can effectively improve the reconstruction rate of given haplotypes.

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.

Thermodynamics of protein folding: methodology, data analysis and interpretation of data.

Thermodynamics of protein folding refers to the stability measurements where structural changes of a given protein in the presence of a denaturing agent are monitored by spectroscopic or calorimetric techniques. In macroscopic point of view, protein stability represents the ratio of the population of its unfolded state to that of folded one in equilibrium condition, while in microscopic point of view, the stability is actually a net value from a combination of favorable and unfavorable contributions that affect the structural integrity of a protein molecule. In this manuscript, the principles and methodological aspects of thermodynamic studies and methods of data analysis as well as interpretation of the results are presented.

Temperature dependent dynamics in highly homologous adenylate kinases.

To correlate the structural features of enzymes to temperature adaptation, we studied psychrophile, mesophile, and thermophile adenylate kinases as model enzymes using bioinformatics and computational tools. Phylogenetic analysis revealed that mesophile and thermophile variants are clustered in one stem of phylogenetic tree and are close to contemporary time, while psychrophile enzyme is more close to their common ancestor. This finding is in good agreement with the process of environmental changes from ice age toward current warm conditions on the earth. We also performed Molecular Dynamics simulation at corresponding temperatures of all enzyme variants including 308, 318, and 328 K. It was found that mesophile enzyme has no distinct deviation of Root Mean Square Deviation (RMSD) and Radius of Gyration (Rg) values from equilibrium states at operating temperature of thermophile enzyme as well as its own optimum temperature. However, psychrophile enzyme undergoes more fluctuations with higher amplitude of change; particularly at 328 K. It was also found that initial increasing of RMSD and Rg for Psychrophile enzyme at all temperatures is occurred gradually; while, the increment of this structural parameters for thermophile enzyme at 328 K is occurred in a highly cooperative and switching manner demonstrating snap structural change of thermophile enzyme in its own temperature. By analysis of Root Mean Square Fluctuation values at different temperatures, we identified two flexible fragments in adenylate kinases so that different dynamic behavior of these regions in mesophile enzyme against operating temperatures of psychrophile and thermophile variants is critical in compensation of flexibility challenges at respective temperatures.

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.

Structural and functional consequences of EF-hand I recovery in mnemiopsin 2.

Mnemiopsin 2 from Mnemiopsis leidyi is a calcium-regulated photoprotein which has luminescence properties in the presence of calcium and coelenterazine. All calcium-regulated photoproteins contain EF-hand loops consisting of 12 individual residues in which the 6th position is occupied by Gly. However, the 6th residue in mneniopsin 2 is Glu rather than Gly. Here, we investigated the structural and functional consequences of substitution of Glu by Gly (E50G variant) using site-directed mutagenesis and spectroscopic procedures. It was revealed that the luminescence activity of the variant was about 17 times greater than that of wild-type (WT) photoprotein. In comparison with WT protein, our variant showed higher optimum temperature and calcium sensitivity as well as slower rate of luminescence decay. Homology modeling and sequence analysis with other known photoproteins showed that EF-hand I loop can affect the luminescence activity of E50G variant. Structural studies using circular dichroism and fluorescence spectroscopy revealed that mutation leads to the reduction in secondary structural content and local structural alterations. Finally, it can be concluded that the activity of E50G variant increases as a result of more flexibility that brought about by Gly essential for adopting the correct conformation for functional activity.

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.

Distribution of dipeptides in different protein structural classes: an effort to find new similarities.

Finding any regularity in the sequences of proteins and determining their correlation with structural features are of great interest for an understanding of molecular biology. We statistically analyzed the relative frequencies of all 400 possible dipeptides in a data set containing randomly selected proteins of different defined structural classes including all-alpha, all-beta, alpha + beta and alpha/beta families. We found that the distribution of dipeptides is not the same for different structural classes, and some of them are significantly far from a random distribution. A tendency of a given amino acid to localize in the first or second position of a dipeptide depending on the structural class of protein was also found. Interestingly, some amino acids may be substituted for each other in the first or second positions of specific dipeptides in each structural class. This finding apparently contrasts with the routine expectation from the viewpoint of amino acid properties, as classically understood.