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Macromolecular interactions in vitro, comparing classical and novel approaches

European Biophysics Journal volume 50, pages 313–330 (2021)Cite this article

A Correction to this article was published on 27 April 2021

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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.

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Acknowledgments

We thank members of Philippe Minard’s and Jean-Baptiste Charbonnier’s teams at I2BC for the sample preparation, Bruno Baron and Bertrand Raynal from Institut Pasteur, Paris for all their expert advices in molecular scale biophysics, and Eric Ennifar from Institut de Biologie Moléculaire et Cellulaire, Strasbourg for sharing its expertise in the study of biomolecular machineries using biophysical approaches. Friederike Möller and Hanna Müller-Landau from Dynamic Biosensors, Aymeric Audfray from Malvern Panalytical, Mathilde Belnou from NanoTemper technologies, and Stephanie Bourgeois and coworkers from Fluidic Analytics for their availability and all the fruitful discussion. We kindly thank all the participants to the MoSBio Training School, all the sponsors without whom this successful event had not been possible, and finally the keynote speakers, Julie Ménétrey and Terence Strick who shared their projects with us. Most of preparatory experiments were performed in the I2BC, PIM platform (https://www.pluginlabs-universiteparissaclay.fr/fr/results/keywords/PIM), while some others were performed in Institut Pasteur, PFBMI platform. Finally, we thank ARBRE-MOBIEU network for its support to publish this article.

Funding

JBC is supported by ARC program (SLS220120605310), French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05, ANR-18-CE44-0008, ANR-20-CE11-0026, INCA 2016-1-PL BIO-11 and INCA SLX4 INCA 2016-159.

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Author notes

  1. Christophe Velours and Magali Aumont-Nicaise contributed equally to this study.

Authors and Affiliations

  1. Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France

    Christophe Velours, Magali Aumont-Nicaise, Jean-Baptiste Charbonnier & Paloma Fernández Varela

  2. Microbiologie Fondamentale et Pathogénicité, MFP CNRS UMR 5234, University of Bordeaux, 146 rue Léo Saignat, 33076, Bordeaux, France

    Christophe Velours

  3. Bioorganic Chemistry & Biophysics Core Facility, Max-Planck-Institute of Biochemistry, Martinsried, Germany

    Stephan Uebel

  4. Molecular Biophysics Platform, Institut Pasteur, Paris, France

    Patrick England

  5. Institute of Biocomputation and Physics of Complex Systems (BIFI), Joint Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Universidad de Zaragoza, 50018, Zaragoza, Spain

    Adrian Velazquez-Campoy

  6. Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, 50009, Zaragoza, Spain

    Adrian Velazquez-Campoy

  7. Instituto de Investigación Sanitaria Aragón (IIS Aragón), 50009, Zaragoza, Spain

    Adrian Velazquez-Campoy

  8. Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas y Digestivas (CIBERehd), 28029, Madrid, Spain

    Adrian Velazquez-Campoy

  9. Fundación ARAID, Gobierno de Aragón, 50009, Zaragoza, Spain

    Adrian Velazquez-Campoy

  10. Institut de Biologie de l’Ecole Normale Supérieure (IBENS), Paris, France

    David Stroebel

  11. Architecture et Réactivité de l’ARN, Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire (IBMC) du CNRS, Strasbourg, France

    Guillaume Bec

  12. NanoTemper Technologies GmbH, Munich, Germany

    Pierre Soule

  13. ForteBio-Sartorius, Dourdan, France

    Christophe Quétard

  14. Université Grenoble Alpes, CNRS, CEA, IBS, Grenoble, France

    Christine Ebel

  15. Architecture et Fonction des Macromolécules Biologiques (AFMB), Marseille, France

    Alain Roussel

Authors
  1. Christophe Velours
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  2. Magali Aumont-Nicaise
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  5. Adrian Velazquez-Campoy
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  6. David Stroebel
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  7. Guillaume Bec
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  8. Pierre Soule
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  12. Jean-Baptiste Charbonnier
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  13. Paloma Fernández Varela
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Contributions

PFV, PE, SU, CE, AVC, AR, JBC authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by CV, MAN, SU, PE, AVC, DS, GB, PS, CQ, CE, AR. The first draft of the manuscript was written by PFV and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Paloma Fernández Varela.

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Conflict of interest

The authors declare no competing interest. Pierre Soule (NanoTemper) and Christophe Quétard (FortéBio) helped during the training without commercial interest.

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The original online version of this article was revised: Due to corrections in Figures. In Fig. 2, the size of the subtitles a to g is reduced; In Fig 3b, “1DNA_1Ku70/Ku80” has been changed to “1DNA-1Ku70/Ku80” and In Fig 4, “50µg” has been changed to “50ng” in the MST cell at the bottom of the table.

Special Issue: COST Action CA15126, MOBIEU: Between atom and cell.

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Velours, C., Aumont-Nicaise, M., Uebel, S. et al. Macromolecular interactions in vitro, comparing classical and novel approaches. Eur Biophys J 50, 313–330 (2021). https://doi.org/10.1007/s00249-021-01517-5

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  • DOI: https://doi.org/10.1007/s00249-021-01517-5

Keywords

  • Molecular scale biophysics
  • Macromolecular interactions
  • Artificial binders
  • Double-stranded DNA breaks repair factors