Functional Application Areas

Protein-Protein Interactions

Proteins-protein interactions are central to many biomolecular processes.  These interactions include:

  • Formation and dissociation of multi-subunit  protein complexes (hetero-oligomers or homo-oligomers)
  • Enzymes binding to protein substrates
  • Antibody-antigen interactions
  • Receptor-protein interactions

Knowledge of these interactions is important to understand how proteins 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 peptide, 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-protein interaction. ITC can measure the heat of dissociation of protein complexes.  Titration of an oligomeric protein into buffer results in heat change due to dissociation into protein monomers.

Most drug targets are proteins, and drug discovery involves identifying compounds which can either inhibit or activate the target protein. 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.

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

References

Determination of the monomer-dimer equilibrium of interleukin-8 reveals it is a monomer at physiological concentrations.
Burrows S. D., Doyle M. L., Murphy K. P., Franklin S. G., White J. R., Brooks I., McNulty D. E., Scott M. O., Knutson J. R., Porter D.
Biochemistry 33, 12741-12745 (1994)

Dissecting homo-heptamer thermodynamics by isothermal titration calorimetry: entropy-driven assembly of co-chaperonin protein 10.
Luke K., Apiyo D., and Wittung-Stafshede P.
Biophys J 89, 3332-3336 (2005)

Characterization of protein-protein interactions by isothermal titration calorimetry.
Velazquez-Campoy A., Leavitt S. A., and Freire E.
Methods Mol Biol 261, 35-54 (2004)

ITC – Protein-Protein Interactions Reference List

ITC – Drug Discovery and Design Reference List

ITC – Protein Engineering and Mutagenesis Reference List

ITC – Protein Subunit Association and Disassociation Reference List

Binding Studies and Differential Scanning Calorimetry (DSC)

Differential Scanning Calorimetry (DSC) is primarily used to characterize biomolecule stability and folding.  When two proteins bind, a Tm of the protein-protein complex is higher than that of a protein alone.  Differential Scanning Calorimetry (DSC) is used to measure the binding constants from Tm shifts due to protein binding to a protein. The binding constant can be estimated from the Tms in the presence and absence of ligand. This method can estimate binding constants up to 1020 M-1.  It can be used for ligands with ultratight binding constants that cannot be measured by other methods.

References

Study of strong to ultratight protein interactions using differential scanning calorimetry.
Brandts, J.F., Lin, L.-N.
Biochemistry 29, 6927-6940 (1990)

An intermediate pH unfolding transition abrogates the ability of IgE to interact with its high affinity receptor FceRIa.
Demarest, S.J., Hopp, J., et al.
J. Biol. Chem. 281, 30755-30767 (2006)

An autosampling differential scanning calorimeter instrument for studying molecular interactions.
Plotnikov, V., Rochalski, A., Brandts, M., Brandts, J.F., Williston, S., Frasca, V., Lin, L.-N.
Assay Drug Devel. Technol. 1, 83-90 (2002)

Need more information? Contact us.