Reconstitution of Isotopically Labeled Ribosomal Protein L29 in the 50S Large Ribosomal Subunit for Solution-State and Solid-State NMR

  • Emeline Barbet-Massin
  • Eli van der Sluis
  • Joanna Musial
  • Roland Beckmann
  • Bernd Reif
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1764)

Abstract

Solid-state nuclear magnetic resonance (NMR) has recently emerged as a method of choice to study structural and dynamic properties of large biomolecular complexes at atomic resolution. Indeed, recent technological and methodological developments have enabled the study of ever more complex systems in the solid-state. However, to explore multicomponent protein complexes by NMR, specific labeling schemes need to be developed that are dependent on the biological question to be answered. We show here how to reconstitute an isotopically labeled protein within the unlabeled 50S or 70S ribosomal subunit. In particular, we focus on the 63-residue ribosomal protein L29 (~7 kDa), which is located at the exit of the tunnel of the large 50S ribosomal subunit (~1.5 MDa). The aim of this work is the preparation of a suitable sample to investigate allosteric conformational changes in a ribosomal protein that are induced by the nascent polypeptide chain and that trigger the interaction with different chaperones (e.g., trigger factor or SRP).

Key words

Isotope labeling Protein complex reconstitution MAS solid-state NMR spectroscopy 

Notes

Acknowledgment

We acknowledge support from the Helmholtz-Gemeinschaft and the Deutsche Forschungsgemeinschaft (Grants Re1435 and SFB-1035, project B07). In addition, we are grateful to the Center for Integrated Protein Science Munich (CIPS-M) for the financial support. We acknowledge support from EMBO (Fellowship ALTF 52-2014) and from the European Commission (EMBOCOFUND2012, GA-2012-600394) and Marie Curie Actions.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Emeline Barbet-Massin
    • 1
    • 2
  • Eli van der Sluis
    • 3
    • 4
  • Joanna Musial
    • 3
  • Roland Beckmann
    • 3
  • Bernd Reif
    • 1
    • 5
  1. 1.Munich Center for Integrated Protein Science (CIPS-M) at Department ChemieTechnische Universität München (TUM)GarchingGermany
  2. 2.Dynamic BiosensorsPlaneggGermany
  3. 3.Gene Center, Department of Biochemistry and Center for Integrated Protein Science Munich (CiPSM)Ludwig-Maximilians-Universität MünchenMunichGermany
  4. 4.Department of Bionanoscience, Faculty of Applied SciencesTU DelftDelftThe Netherlands
  5. 5.Deutsches Forschungszentrum für Gesundheit und UmweltHelmholtz-Zentrum München (HMGU)NeuherbergGermany

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