Functional Application Areas

Interactions with Proteins

Proteins are involved in essentially all biological processes: enzymes catalyze chemical reactions, protein receptors are involved in signaling and transport, and antibodies are vital to the immune response.  As part of their function, these proteins specifically bind a ligand such as a substrate, inhibitor, cofactor, carbohydrate or a large biomolecule - a lipid, nucleic acid or another protein. Proteins are often engineered, so there must be assays that can be used with a wide range of affinities. Understanding how proteins function and interact with other molecules is key in biochemistry and drug discovery and development.

Accompanying the rapid advances in structural biology is the definition of biochemical function and mechanism in terms of molecular forces.  However, knowledge of protein structure alone does not ensure accurate prediction of function and biological activity. The complete characterization of any binding interaction requires a quantification of the binding affinity, number of binding sites, and the thermodynamics. 

Thermodynamics provides information on the energetic forces that drive biomolecular interactions. Enthalpy (ΔH) and entropy (ΔS), reveals the forces that drive complex formation and mechanism of action, and provides information on conformational changes, hydrogen bonding, hydrophobic interactions, and charge-charge interactions.  This information is used to describe the function and mechanism at a molecular level. 

Isothermal Titration Calorimetry (ITC) is a powerful analytical tool which measures the binding affinity and thermodynamics between any two biomolecules. ITC is considered the “gold standard” assay for binding

ITC is vital in the study of multi-probe structure activity relationships (SAR) since it can detect contributions that affinity-only methods may miss.  For example, the affinity measured by these methods may be similar for a wild-type and mutant protein binding to a drug, but ITC can reveal differences in ΔH and ΔS that can describe the mechanism of action of binding.  This information can validate in-silico modeling. ITC is commonly used to validate other binding assays.

ITC is also becoming an important tool in characterizing drug-target interactions, and can be used in many different stages of Drug Discovery and Development. 

ITC can be used to characterize any binding event with a protein, including:

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