*3.6. Principal Component Analysis and Free Energy Landscape*

Principal component analysis (PCA) is a comprehensive and useful tool to investigate conformational changes of proteins [29]. The theoretical notes on PCA have been explained in various preceding reports [29,31,48]. To realistically discover the variations in the structural configuration of HSA and the HSA–HpzA docked complex, PCA was employed on the equilibrized trajectories from the MD study. The PCA was performed using the GROMACS utilities by calculating the eigenvalues (EVs) and their projection along the two principal components (PC1 and PC2). FEL analysis has been widely used in examining the folding mechanisms and overall stability of protein and protein–ligand complexes [49]. FELs for HSA and the HSA–HpzA docked complex were generated through the *gmx sham* tool of the GROMACS package.
