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).
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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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Barbet-Massin, E., van der Sluis, E., Musial, J., Beckmann, R., Reif, B. (2018). Reconstitution of Isotopically Labeled Ribosomal Protein L29 in the 50S Large Ribosomal Subunit for Solution-State and Solid-State NMR. In: Marsh, J. (eds) Protein Complex Assembly. Methods in Molecular Biology, vol 1764. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7759-8_6
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DOI: https://doi.org/10.1007/978-1-4939-7759-8_6
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