Manipulation of ligand binding affinity by exploitation of conformational coupling

Traditional approaches for increasing the affinity of a protein for its lig and focus on constructing improved surface complementarity in the complex b y altering the protein binding site to better fit the ligand. Here we prese nt a novel strategy that leaves the binding site intact, while residues tha t allosterically affect binding are mutated. This method takes advantage of conformationally distinct states, each with different ligand-binding affin ities, and manipulates the equilibria between these conformations. We demon strate this approach in the Escherichia coli maltose binding protein by int roducing mutations, located at some distance from the ligand binding pocket , that sterically affect the equilibrium between an open, apo-state and a c losed, ligand-bound state. A family of 20 variants was generated with affin ities ranging from a similar to 100-fold improvement (7.4 nM) to a similar to two-fold weakening (1.8 mM) relative to the wild type protein (800 nM).