Functional Application Areas

Protein-Carbohydrate Interactions

Lectins and other carbohydrate binding proteins are found in animals, plants and bacteria. Carbohydrate binding proteins have been shown to be involved in many cellular processes including:

  • Trafficking and clearing glycoproteins
  • Cell adhesion
  • Digestion
  • Immune response
  • Glycosylation
  • Cell recognition
  • Tumor genesis
  • Apoptosis
  • Heparin binding proteins
  • Endotoxin binding

Knowledge of these protein-carbohydrate interactions is important to understand how these biomolecules function in biological systems. There have been rapid advances in structural biology and relating structure to biochemical function and mechanism. However, knowledge of protein and carbohydrate 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 sugar, 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 carbohydrates binding a protein), binding of inhibitors of protein-carbohydrate interactions, and allosteric effects of ligands to the protein-carbohydrate interactions.

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

Thermodynamic studies of lectin-carbohydrate interactions by isothermal titration calorimetry.
Dam T. K. and Brewer C. F.
Chem Rev 102, 387-429 (2002)

Multivalent protein-carbohydrate interactions: isothermal titration microcalorimetry studies.
Dam T. K. and Brewer C. F.
Methods Enzymol 379, 107-128 (2004)

ITC – Protein-Carbohydrate Interactions Reference List

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