Improving Protein Stability: Identifying Suboptimal Residues for Evolutionary Enhancement

The energy difference between the improved structure and the single point mutant was calculated using the ligand-free D-Lac structure as the input file. Positions with multiple mutations in ΔΔG≤0 R.e.u. were marked as suboptimal, and pSUFER was accessible at (https://psufer.weizmann.ac.il). Residues near the binding pocket that were repeatedly labeled as suboptimal were selected as candidate residues for evolution to enhance the stability of the fold.