Checking the quality of the model produced
Employing four servers including ProSA-web, QMEAN, RAMPAGE, and Verify 3D, the best model was selected before and after mutation. The overall model quality was additionally examined using ProSA-web along with QMEAN servers. Then, the standardized score was calculated, which is a total model quality index. The results of PorSA-web and QMEAN Z-score indicated that the models have desirable qualities. In addition, 81.52% of amino acids having a score of 3D-1D> = 0.2 in verify 3D and RAMPAGE with 98.9% residues in the allowed region. The wild-type and E121D mutant were visualized using PyMOL as shown in Figure 2A-B.
The hydrogen bonding results analysis in Glu, Asp 121 in wild-type and mutant protein using PyMOL software
The results showed that glutamate has four hydrogen bonds in the side chain with the surrounding molecules in wild-type. However, there is an extra hydrogen bond that binds the aspartate to Zn1388 of chain B in E121D mutation (Fig.3A-B).
Analysis of the results of the distance between Zn 1388 of chain B and Cu1156 of RCY to glutamate 121 and aspartate 121 (after mutation) chain A using PyMOL software.
In E121D mutation, an increase occurred in the distance between Zn1388 of chain B and aspartate 121 of chain A compared to glutamate 121 (2.2 angstroms in E121 and 4.4 in D121). Also, a decrease occurred in the distance between aspartate 121 of chain A and Cu1156 of RCY at RCY/Cytochrome c552 complex (16.9 angstroms in E121 and 15.3 in D121) (Fig. 4A-B).
The results of MD simulation analysis and effects mutation on stability and protein secondary structure
MD simulation was performed to study the configuration, and stability in the wild-type protein and the changes of these parameters in the mutant protein, for both types of proteins. In addition, MD simulation is considered a useful method to support the experimental results in protein mutations through structural specifications at the atomic level . RMSD, RMSF, SASA, H-bonds, Rg, and DSSP were analyzed throughout the simulation time (Table 2, 3).
To find out the effect of the mutation on conformational changes and the stability of the protein structure, the RMSD of Cα atoms was calculated for both the wild-type and mutant compared to the original structure . The RMSD plot of the wild and mutant structures indicated the convergence pattern during 100 ns simulations (Fig. 5A). Convergence obtained for wild and mutant structures confirmed that the mutant protein has less sustainability than the wild-type.
The RMSF of each residue was measured to assign the mutation effect on the residues' dynamic behavior in wild and mutant proteins . Overall, the mutant protein had more flexibility (RMSF) than the wild-type (+0.0002 increase) . The RMSF plan is plotted for wild and mutant structures, as shown in Figure 5B.
Intermolecular hydrogen bonding is the electrostatic force between two polar bonds. In this bonding, hydrogen is bonded covalently to the highly electronegative atoms like nitrogen and oxygen. H bond was calculated for wild and mutant types. The average number of H bonds for the mutated structure was 125.78 ± 4.52 and -2 number decreased compared to wild protein (Table 2). The difference in the mutant H bonds confirmed the flexibility of the mutant structure (Fig. 5C).
Rg determines the compression of the protein. As the radius of gyration is higher, the protein tends to be more unstable, unfolding, and low compact . The values of Rg for mutated variants were 1.62 ± 0.001 nm, with a slight change (+0.01 increase) in protein folding and its compactness (Table 2) (Fig. 5D).
SASA indicates the level of protein access to the solvent . The change in SASA measured the accessibility of the protein to the solvent in the wild and mutant structure during simulations (Fig. 5E). The average of SASA for the mutated structure was 103.10 ± 0.59 nm2 and +1.23 increased compared to wild protein (Table 2).
Figure 6 shows the alterations in the secondary structure of the protein during the simulations. The DSSP scheme has been used to calculate the secondary structure of wild-type and mutant protein. The analysis of wild and mutant structures shows the number of amino acids involved in different structures and the increase or decrease in the number of amino acids in the structures. The most changes are related to the turn structure with an increase of 2.27 amino acids, and the α helix structure with a decrease of 1.27 amino acids. The bar graph of the secondary structure for wild-type and E121D mutant systems is shown in Figure 6A. The amount of Structure, coil, Turn, and beta bridge in the mutant protein has increased but, beta-sheet, alpha-helix, have diminished. The highest value is shown in the secondary structure of the two systems for coil and alpha-helix. And the largest change is related to the turn structure (1.24% increase), which is shown in Table 3.
Results of φ and ψ angle analysis of amino acids in wild and mutant proteins by Ramachandran method
The results were reported as diagrams of changes in angles according to time, to investigate the state of the protein at the mutation point. According to the angles φ and ψ before and after mutation using the Ramachandran method, it was found that the amino acid studied did not change significantly in terms of angles φ and ψ (Fig. 7, Table 4).
The Wild and mutant proteins Ramachandran scheme produced by PROCHECK
To compare the Ramachandran results of simulated wild and mutant protein (E121D), the pdb files were uploaded and the results are shown in Figure 8 and Table 5. Before the mutation, 95.5% of the amino acids are present in most favored regions, which after the mutation, its amount has decreased to 84%. There were no amino acids in the allowed and disallowed regions before the mutation, but after the E121D mutation, two of the amino acids (alanine 9, histidine 54) were in the allowed regions.