Table 3
3.6 MD simulations of WT and M3
For investigating the structural stability of the “best” mutant M3 with improved organic solvents stability, MD simulations of WT and M3 were performed at 308 K for 20 ns. The parameter of Root-mean-square deviation (RMSD) is used for exploring the fluctuation of protein conformation, and protein stability is negatively correlated with its RMSD values.[35] As shown in Figure 5A, C, E, and G, M3 reached its equilibrium earlier than WT under different concentrations of DMSO, and the overall RMSD values of WT were higher than M3. The average RMSD values for WT were 1.733, 1.917, 1.922, and 1.981 Å, whereas the values for M3 declined to 1.521, 1.557, 1.598, and 1.603 Å. These results indicated that mutant M3 was more rigid than the WT under different concentrations of DMSO (ranging from 15%~45%, v/v ) at 308 K. The results of Root-mean-square fluctuations (RMSF) were presented in Figure 5B, D, F, and H, representing the degree of freedom of each atom in a protein, and the residues with larger RMSF values reflected more degrees of freedom than those with smaller RMSF values.[35] The overall RMSF plots of WT at different concentrations of DMSO were matched with M3. However, the regions of the mutation sites had apparent changes. The RMSF values of the N-terminal loop and C-terminal loop were decreased from WT to M3. The residues 23~30, 198~200, and 258~260 in WT had higher RMSF values than M3 (Table S8). And the average RMSF values of the overall structures decreased from 1.147, 1.180, 1.063, and 1.021 Å on WT to 1.121, 1.097, 0.972, and 0.982 Å on M3 under 15%, 25%, 35% and 45% DMSO, respectively. The reduction of regional and global amino acid RMSF values indicated that the mutant M3 had lower flexibility, which would significantly contribute to its organic solvent stability in different concentrations of DMSO.