A Biophysical Toolkit for Molecular Interactions

Abstract

Intermolecular interactions inside and around the cell are key to maintain basic cell homeostasis and to sense and react to external stimuli. Moreover, the action of therapeutics and other xenobiotics occurs through physical interactions of these agents with biomolecules present in the cell or tissue, thereby interfering with the ‘native’ interactions of the biomolecules. Biophysical characterisation of molecular interactions is a bottom-up reductionist approach that implies measuring the physicochemical properties of the interacting species (two biomacromolecules or a biomacromolecule and a small-molecule ligand) in order to quantify the binding strength (thermodynamics) and association and dissociation speed (kinetics) between them. A large variety of macroscopic experimental techniques are available and constitute a biophysical toolkit to study molecular interactions in vitro. Among others, isothermal titration calorimetry, surface plasmon resonance, analytical ultracentrifugation, nuclear magnetic resonance, microscale thermophoresis, thermal shift and fluorescence-based methods are most commonly used.

Key Concepts

• The strength (affinity) and speed (kinetics) of interactions between biomolecules can be quantified using biophysical experimental methods.
• Isothermal titration calorimetry (ITC), fluorescence and nuclear magnetic resonance (NMR) spectroscopies, thermal shift assay (TSA), analytical ultracentrifugation (AUC), microscale thermophoresis (MST) and surface plasmon resonance (SPR) are arguably the most used and versatile techniques to study binding in vitro and constitute a biophysical toolkit.
• Equilibrium affinity constants can be robustly obtained by titration using ITC, fluorescence, NMR, MST, AUC and SPR.
• Kinetic information can be obtained from time-resolved fluorescence spectroscopy and SPR methods. The recently developed kinITC method allows obtaining kinetic information from ITC data.
• Biophysical techniques can tackle the study of a wide range of systems and great progress has been made in the characterisation of interactions taking place at the cell membrane.
• Basically, all biophysical methods can be automatised to screen for small-molecule ligands of proteins and other macromolecules, providing starting points for chemical biology and drug discovery.