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FRENCH INFRASTRUCTURE FOR INTEGRATED STRUCTURAL BIOLOGY

Calorimetry.jpg

CBS

CALORIMETRY

Biological phenomena in general involve molecular recognition on an intramolecular or intermolecular level. Intermolecular processes involve interaction between different types of macromolecules or between a macromolecule and small ligands.

Understanding the interplay between a macromolecular structure and the energetics of its stability and the binding of its partners relies on the knowledge of the parameters describing the equilibrium constant of the free energy, as a sum of enthalpy and entropy.

Modern calorimeters permit direct determination of enthalpy values for binding reactions and conformational transitions in biomolecules. Complete thermodynamics of free energy, enthalpy, and entropy are obtained for reactions of interest in a straightforward manner. Such data are of enormous value in drug design where they provide information about the balance of driving forces.

At the CBS, the Isothermal titration calorimetry (ITC) is used to investigate all types of protein interactions, with an emphasis on protein-small molecule interactions.

Device: Microcal VP ITC

Thermal shift assay is a thermodenaturation assay monitoring the thermal stability of proteins and investigate factors affecting this stability. This rapid and simple technique can be used in medium-throughput mode to screen optimal solubility conditions for purified macromolecules.

At the CBS, the differential scanning fluorimetry (DSF) method is available.

The optimization of proteins solubility and stability properties improves the success rate of their structural studies [1]. Changes in the thermal stability of the protein–ligand or protein-peptide complexes relative to the stability of the protein alone allow to rapidly identify promising complexes for further structural characterization and to assign functions.

Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination Vedadi et al.,Proc Natl Acad Sci. (2006) , 103, 15835-40.

Device:

Mx3005P QPCR system.