THE RELATIONSHIP BETWEEN LIGAND-BINDING THERMODYNAMICS AND PROTEIN-LIGAND INTERACTION FORCES MEASURED BY ATOMIC-FORCE MICROSCOPY

The interaction forces between biotin and a set of streptavidin site-d irected mutants with altered biotin-binding equilibrium and activation thermodynamics have been measured by atomic force microscopy. The AFM technique readily discriminates differences in interaction force betw een the site-directed (Trp to Phe or Ala) mutants. The interaction for ce is poorly correlated with both the equilibrium free energy of bioti n binding and the activation free energy barrier to dissociation of th e biotin-streptavidin complex. The interaction force is generally well correlated with the equilibrium biotin-binding enthalpy as well as th e enthalpic activation barrier, but in the one mutant where these two parameters are altered in opposite directions, the interaction force i s clearly correlated with the activation enthalpy of dissociation. The se results suggest that the AFM force measurements directly probe the enthalpic activation barrier to ligand dissociation.