Study of interactions in protein samples

Determination of protein-protein, protein-DNA, or protein-ligand interactions

Different instruments are available at the CBS to measure interactions with proteins.
VP-ITC microcalorimetry is based on the measurement of the heat emitted or absorbed during a reaction of interaction between molecules or during changes in conformation such as the folding of proteins. Isothermal Calorimetric Titration (ITC) is used to study the factors influencing interactions between molecules. It is possible to determine the equilibrium constant (Ka) as well as the stoichiometry of the interaction. It also gives access to the thermodynamic constants characterizing the interaction such as the variations of the enthalpy (H) and the entropy (S).
The main applications of this technique are:

• Determination of affinity constants in protein/protein or protein/ligand or protein/DNA interactions.
• Determination of the stoichiometry of the biochemical interaction.

SEC-MALLS technique can also be used to study macromolecular complexes. It allows to define the absolute molecular mass of the protein or complex analyzed regardless their size and conformation. It includes exclusion chromatography (SEC) coupled online with a measurement of UV absorbance, laser light scattering (LS) and refractive index (RI). The proteins are separated by exclusion chromatography, the light scattered by the proteins is directly proportional to their molecular weight and their concentration. This technique also makes it possible to determine stoichiometry in a complex.
The Thermal Shift Assay (TSA) technique measures the denaturation temperature variation in a protein sample during a rapid temperature gradient in function of experimental conditions. The fluorescent dye Sypro Orange is used to measure the exposure of the protein hydrophobic regions during its denaturation. Like that, TSA can be used to screen potential interactions between a protein and different ligands (peptides, chemical library).

Example of interaction measurement between a protein of interest and a synthetic ligand with VP-ITC MicroCal

VP ITC Microcalorimetry

SEC- MALS

Thermal Shift Assay