3.3 Molecular Dynamics Simulation
The molecular dynamic method was used to analyze the physical movements
of atoms and molecules and to study conformational change at the atomic
level. To assess the binding stability and determine binding-free energy
against EGFR active site, three scaffolds and scFv were subjected to 50
ns molecular dynamic simulation. Further, all four scaffolds were
analyzed by RMSD, RMSF, H bond, and MMPBSA calculation to examine the
protein stability and dynamic behavior throughout the simulation period.
The variation of the all scaffolds-receptor complex was determined by
the root mean square deviation (RMSD) during the 50 ns MD simulation.
RMSD of alpha carbon was calculated (Fig.2), and results indicated that
all four complexes remained stable throughout the simulation with an
average RMSD 0.75, 2.65, 0.2, 2.7 for scFv (Orange), Scaf1 (Gray), Scaf2
(Blue), and Scaf3 (Red), respectively. A slight fluctuation in the case
of the scFv complex was noted during the first 6 ns, which achieved the
equilibrium and remained stable throughout the simulation. Measuring the
average movement of the atom position at the specific temperature and
pressure was performed by root mean square fluctuation (RMSF) analysis.
The fluctuations in the constituents’ residues were observed for all
four structures and plotted to compare the flexibility of each residue
in complexes (Fig.3). RMSF was calculated for EGFR DIII, SCFV, and three
selected scaffolds. Low RMSF values obtained for all four complexes
indicate good stability of the system. Although there are little
fluctuations in some residues of the scFv and Scaf2, all grafted
residues in designed scaffolds had low RMSF values. Otherwise, the
fluctuation during all four CDR loops-receptor interactions was below
0.2 nm, which is perfectly acceptable and shows good stability of
designed structures.