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.