Stability of Ligand-induced Protein Conformation Influences Affinity in Maltose-binding Protein

•Thermodynamic framework for engineering the ligand-binding affinity of proteins.•Free energy difference between ligand-free and -bound states affects affinity.•smFRET analysis of mutations in the periphery and outside the ligand-binding site.•Affinity is tuned by differentially altering stability of different conformations.