Functional Application Areas

Binding

Binding Studies

All cellular processes require specific binding and molecular recognition between biomolecules. Knowledge of these interactions is important to understand how proteins, nucleic acids and lipids function in biological systems. There have been rapid advances in structural biology and relating structure to biochemical function and mechanism. However, knowledge of 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 can be used to describe the function and mechanism at a molecular level.

Binding Studies with Isothermal Titration Calorimetry (ITC)

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, and has many advantages:

  • Universal assay – directly measures heat change associated with binding
  • Label-free - uses native materials
  • True in-solution technique
  • Requires minimal assay development
  • Has no molecular weight limitations
  • Non-optical
  • Versatile, can be used with any biomolecule - proteins, nucleic acids, small molecule drugs, lipids, etc.
  • Can be used with a wide range of biological buffers, ionic strengths, pH’s
  • Has been cited in thousands of publications

In a single ITC experiment, one can determine:

  • Binding affinity - Kd
    • Directly measure sub-millimolar to nanomolar
    • Can extend affinity range to picomolar using competitive ITC method
  • Number of binding sites
  • Multiple and different binding sites
  • Enthalpy (ΔH) and entropy (ΔS) of 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 also commonly used to validate other binding assays.

ITC is becoming an important tool in characterizing drug-target interactions, and can be used in different stages of Drug Discovery and Development. ITC is versatile and can be used to characterize binding between any two molecules.

Binding Studies with Differential Scanning Calorimetry (DSC)

Differential Scanning Calorimetry (DSC) is primarily used to characterize stability and folding of macromolecules such as proteins. When a ligand preferentially binds to the native form of a protein, the protein is stabilized and the Tm of the protein-ligand complex is higher than that of the protein in the absence of ligand. If the ligand preferentially binds to the denatured protein, the Tm decreases in the presence of the ligand. Differential Scanning Calorimetry (DSC) is used to measure the binding constants from Tm shifts due to ligand binding to a protein. The binding constant of the ligand can be estimated from the Tms in the presence and absence of ligand, as long as the concentration of ligand in the DSC cell is known. This method can estimate binding affinities up to 1020 M-1. It can be used for ligands with ultratight binding constants that cannot be measured by other methods, as well as a screening assay for drug discovery.

Need more information? Contact us.