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.