Centre de Biologie Structurale

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

Study of the thermostability of protein samples

Determination of the denaturation temperature of a protein sample

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. The main applications of this technique are:

• Improvement of protein stability conditions (protein purification and conservation) by screening of different buffer conditions (pH, salts, additives).
• Measurement of mutation and deletion effects on protein stability.
• Screening of potential interactions between a protein and different ligands (peptides, chemical library).

Similarly, NanoDSF allows to determine the denaturation temperature of a protein sample. It is based on the fluorescence variation ratio of tryptophan and tyrosine contained in the protein in function of the temperature. The main applications of this technique are:

• Quality control of protein samples.
• Study of protein and peptide thermal stability in the presence or absence of ligands, detergents, additives, etc.
• Screening of conditions of protein preparation and storage.

Example of denaturation curve obtained on a protein of interest with Nanotemper™ Tycho NT.6

Thermal Shift Assay

Nano DSF

Study of protein samples in solution using diffusion of light

Determination of the size and oligomeric state of a protein sample

Dynamic light scattering (DLS) is a non-destructive spectroscopic technique that allows to determine hydrodynamic radius and molar mass of molecules in solution. This method measures the scattering of particles subjected to Brownian motion and calculates the size distribution of the particles using the Stokes-Einstein relationship. The main applications of this technique are:

• Determination of hydrodynamic radius of a protein from which the apparent molecular weight can be deduced (if the protein can be considered as a globular particle).
• Knowledge of molecule folding state.
• Checking of sample homogeneity by determining the state of mono- or poly-dispersity.

The SEC-MALS technique allows to define the absolute molecular mass of the protein or complex analyzed regardless their apparent 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.
The main applications of this technique are:

• Determination of molecular weight of proteins, protein/protein complexes, proteins/nucleic acids, nanodisks, etc.
• Determination of protein oligomerization state and stoichiometry of complexes.

Example of elution profile with the deduced molar mass of a protein of interest obtained with MALS Wyatt

Dynamic Light Scattering (DLS)

SEC- MALS

Study of interaction kinetics and enzymatic pathways

Determination of the transient kinetics of protein-ligand interactions and of enzymatic processes

Transient kinetics techniques (stopped-flow, quench-flow, cryoenzymology) are used to measure enzymatic kinetics on the millisecond time scale. The main applications of these techniques are :

• Protein-ligand interaction measurements: k_on, k_off, K_d.
• Determination of reaction mechanisms: rate constants of each step, determination of the kinetically limiting step.
• Characterization of enzymatic inhibitors: type of inhibition, K_i.

Example of transient kinetics obtained with quench-flow and relationship between the transient phases and different steps of the reaction pathway.

Stopped-flow

Quench-flow

Cryo Quench-flow