Abstract
Biophysical quantification of protein interactions is central to unveil the molecular mechanisms of cellular processes. Researchers can choose from a wide panel of biophysical methods that quantify molecular interactions in different ways, including both classical and more novel techniques. We report the outcome of an ARBRE-MOBIEU training school held in June 2019 in Gif-sur-Yvette, France ( https://mosbio.sciencesconf.org/ ). Twenty European students benefited from a week's training with theoretical and practical sessions in six complementary approaches: (1) analytical ultracentrifugation with or without a fluorescence detector system (AUC-FDS), (2) isothermal titration calorimetry (ITC), (3) size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), (4) bio-layer interferometry (BLI), (5) microscale thermophoresis (MST) and, (6) switchSENSE. They implemented all these methods on two examples of macromolecular interactions with nanomolar affinity: first, a protein-protein interaction between an artificial alphaRep binder, and its target protein, also an alphaRep; second, a protein-DNA interaction between a DNA repair complex, Ku70/Ku80 (hereafter called Ku), and its cognate DNA ligand. We report the approaches used to analyze the two systems under study and thereby showcase application of each of the six techniques. The workshop provided students with improved understanding of the advantages and limitations of different methods, enabling future choices concerning approaches that are most relevant or informative for specific kinds of sample and interaction.
Highlights
Macromolecular interactions play a central role in the activation/inactivation of most cellular mechanisms
Biophysical measurements of alphaRep interactions We characterized the interaction of A3 with its binders Rep2 and Rep17 using the following techniques presented in the Fig. 1: in solution approaches, which do not require labeling (SECMALS and Isothermal Titration Calorimetry (ITC)), in solution approaches that require labeled protein or DNA (MST and AUCFDS), and surface approaches (BLI and switchSENSE)
AlphaRep interactions measured by label free in solution approaches
Summary
Macromolecular interactions play a central role in the activation/inactivation of most cellular mechanisms These interactions can be measured in cellulo, or in vitro, and predicted in silico. The classical in cellulo methods (such as tap-tag or two-hybrid) allow large-scale studies, but in order to confirm that a direct interaction occurs between two macromolecules, quantitative in vitro measurements are needed. These measurements allow to characterize interactions in terms of affinity, and to determine additional kinetic and thermodynamic parameters, as well as to define the hydrophobic or hydrophilic nature of the interface. By in silico docking analysis, we can predict the structure of macromolecular complexes or the impact of functional substitutions helping to optimize experimental design (Andreani and Guerois, 2014)
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