Experimental evidence indicates that the primary structure of the β2-α2 loop region (residues 165-175) in mammalian prion proteins (PrP) influences the conversion from the cellular species (PrP^C) to the β-sheet-rich aggregate. Here, we captured the transition of the β2-α2 loop from 3₁₀-helical turn to β turn by unbiased molecular dynamics simulations of the single-point mutant Y169G. Multiple conformations along the spontaneous transition of the mutant were then used as starting point for sampling of the free-energy surface of the wild type and other single-point mutants. Using two different methods for the determination of free energy profiles, we found that the barrier for the 3₁₀-helical turn to β turn transition of the wild type is higher by about 2.5 kcal/mol than for the Y169G mutant, which is due to favorable stacking of the aromatic rings of Y169 and F175, and a stable hydrogen bond between the side chains of Y169 and D178. The transition of the β2-α2 loop to β turn increases the solvent-exposure of the hydrophobic stretch 169-YSNQNNF-175. The simulations indicate that the strictly conserved Y169 in mammalian prion proteins stabilizes the 3₁₀-helical turn in the β2-α2 loop, thus hindering the conversion to an aggregation-prone conformation.