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A strategy for co-translational folding studies of ribosome-bound nascent chain complexes using NMR spectroscopy

Nature Protocols volume 11, pages 14921507 (2016) | Download Citation

Abstract

During biosynthesis on the ribosome, an elongating nascent polypeptide chain can begin to fold, in a process that is central to all living systems. Detailed structural studies of co-translational protein folding are now beginning to emerge; such studies were previously limited, at least in part, by the inherently dynamic nature of emerging nascent chains, which precluded most structural techniques. NMR spectroscopy is able to provide atomic-resolution information for ribosome–nascent chain complexes (RNCs), but it requires large quantities (≥10 mg) of homogeneous, isotopically labeled RNCs. Further challenges include limited sample working concentration and stability of the RNC sample (which contribute to weak NMR signals) and resonance broadening caused by attachment to the large (2.4-MDa) ribosomal complex. Here, we present a strategy to generate isotopically labeled RNCs in Escherichia coli that are suitable for NMR studies. Uniform translational arrest of the nascent chains is achieved using a stalling motif, and isotopically labeled RNCs are produced at high yield using high-cell-density E. coli growth conditions. Homogeneous RNCs are isolated by combining metal affinity chromatography (to isolate ribosome-bound species) with sucrose density centrifugation (to recover intact 70S monosomes). Sensitivity-optimized NMR spectroscopy is then applied to the RNCs, combined with a suite of parallel NMR and biochemical analyses to cross-validate their integrity, including RNC-optimized NMR diffusion measurements to report on ribosome attachment in situ. Comparative NMR studies of RNCs with the analogous isolated proteins permit a high-resolution description of the structure and dynamics of a nascent chain during its progressive biosynthesis on the ribosome.

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Acknowledgements

We thank Bernd Bukau (Ruprecht-Karls-Universität Heidelberg) for anti-SecM antibodies. We acknowledge the use of the ISMB Biological NMR Facility, UCL. We thank T. Frenkiel, G. Kelly and A. Oregioni and acknowledge the use of the biomolecular NMR facilities of the MRC NMR Centre at the Francis Crick Institute. This work was supported by a New Investigator Award (BBSRC BBG0156511, to J.C.), a Wellcome Trust Investigator Award (097806/Z/11/Z, to J.C.) and an AlphaOne Foundation grant (to L.D.C.); A.L.R. is an NHMRC CJ Martin Fellow.

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Author notes

    • Anaïs M E Cassaignau
    •  & Hélène M M Launay

    These authors contributed equally to this work.

Affiliations

  1. Institute of Structural and Molecular Biology, University College London and Birkbeck College, University of London, London, UK.

    • Anaïs M E Cassaignau
    • , Hélène M M Launay
    • , Maria-Evangelia Karyadi
    • , Xiaolin Wang
    • , Christopher A Waudby
    • , Annika Deckert
    • , Amy L Robertson
    • , John Christodoulou
    •  & Lisa D Cabrita

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Contributions

A.M.E.C., H.M.M.L., C.A.W., J.C. and L.D.C. designed the study. A.M.E.C., H.M.M.L., M.-E.K., X.W., C.A.W., A.D., A.L.R. and L.D.C. performed the research. A.M.E.C., C.A.W., H.M.M.L., J.C. and L.D.C. wrote the paper. All authors discussed the results and contributed to the final version of the paper.

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The authors declare no competing financial interests.

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Correspondence to John Christodoulou or Lisa D Cabrita.

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https://doi.org/10.1038/nprot.2016.101

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