Transition state thermodynamic analysis using Biacore T 100 Providing information crucial for predicting molecular recognition and structure - based drug design

Biomolecular interactions may be defined at several levels. The simplest quantitative descriptor, affinity, is defined by the affinity constant, KD, a measure of the strength of binding at equilibrium. This term may be further resolved into kinetic descriptors of rate of association (defined by the association rate constant (ka) and dissociation (kd). Kinetic information, however, does not reveal the molecular mechanisms underlying the rates at which a complex forms and dissociates. Although structural aspects of complementarity may be revealed by technologies such as X-ray crystallography or NMR, the character and quantitative contributions of non-covalent forces within the binding site can only be rationalized by studying interaction thermodynamics. Fully understanding molecular recognition by being able to predict binding energetics from the three-dimensional structure of protein complexes through thermodynamic analysis may well provide the basis for structure-based molecular design of drugs and engineered antibodies. Figure 1. Thermodynamic assays on Biacore T100; (1) Samples are injected over a target protein immobilized on a sensor surface. Each interaction is analyzed at several temperatures; (2) Affinity and kinetic rate constants for each interaction are automatically calculated; (3) Software wizards integrate affinity and kinetic rate constants into thermodynamic equations to calculate ∆Go, ∆Ho and ∆So at equilibrium, as well as during complex association and dissociation (∆Go‡, ∆Ho‡ and ∆So‡). Interactions between Mab fragments and immobilized HEL performed at different temperatures within a temperaturecontrolled microenvironment Calculation of affinity and kinetic rate constants