Functional Application Areas

Protein Receptors

Protein receptors are important in many cellular processes, including:

  • Cell transport
  • Ion channels
  • Cell adhesion
  • Histocompatability
  • Cell signaling pathways and transduction
  • Angiogenesis and tumorogenesis

Protein receptors, such as G-protein coupled receptors (GPCRs) are also a major class of drug targets, since they are involved in many different biological pathways.

Protein receptors interact with other proteins, as well as other biomolecules, and knowledge of these interactions is important to understand how receptors function in biological systems. There have been rapid advances in structural biology and relating structure to biochemical function and mechanism. 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 affinity, number of binding sites, and the thermodynamics. 

Thermodynamic data, specifically enthalpy (ΔH) and entropy (ΔS), reveal the forces that drive complex formation and mechanism of action. Thermodynamics provide 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 receptor, 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 also commonly used to validate other binding assays.

ITC is also used to characterize ligand specificity (i.e. a series of peptides binding to the same site of a protein), binding of inhibitors of protein-protein interactions, and allosteric effects of ligands in the protein interactions.

Since ITC is done in-solution, it can utilize any biological buffer, and can use detergents for solubilization of membrane proteins... For a full characterization of a biomolecular interaction, it is important to observe how salt, pH, temperature, etc affects binding affinity and thermodynamics.

Most drug targets are proteins, and drug discovery involves identifying compounds which can either inhibit or activate the target protein. ITC is becoming an important tool in characterizing drug-target interactions, and can be used in different stages of Drug Discovery and Development.

References

Advances in membrane receptor screening and analysis.
Cooper M. A.
J Mol Recognit 17, 286-315 (2004)

Approaches to the description and prediction of the binding affinity of small-molecule ligands to macromolecular receptors.
Gohlke H. and Klebe G.
Angew Chem Int Ed Engl 41, 2644-2676 (2002)

ITC – Protein Receptor Studies Reference List

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