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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 1492–1507 (2016)Cite this article

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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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Figure 1: General scheme for structural studies of RNCs by NMR spectroscopy.
Figure 2: Biochemical assessment of purified RNCs.
Figure 3: NMR analysis of RNC integrity.
Figure 4: Heteronuclear spectra of RNCs and monitoring of RNC integrity.

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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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  1. Anaïs M E Cassaignau and Hélène M M Launay: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Structu,

    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

  2. ral 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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Correspondence to John Christodoulou or Lisa D Cabrita.

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Integrated supplementary information

Supplementary Figure 1 2D correlation spectra of FLN5 in the presence of 70S ribosomes

a SOFAST 2D correlation spectrum overlay of 5 μM 15N-labelled FLN5 in the absence (red) and presence (cyan) of 5 μM 70S ribosomes. b The relative intensities of an equimolar sample of FLN5 and 70S ribosomes (5 µM) vs. FLN5 alone (5 µM), plotted against primary sequence, shows that the intensities of FLN5 resonances remain unchanged in the presence of ribosomes, making it a tractable system for RNC studies. The analogous study can be undertaken with the unfolded polypeptide. All uncertainties have been derived from the spectral noise. Adapted from ref. 1 with permission from Nature Publishing Group.

Supplementary Figure 2 Assessment of ribosome background labelling

To assess the level of ribosomal background labelling within RNC samples, 15N/13C filtered 1H 1D (red) and 15N/13C edited 1H 1D (grey) spectra are collected. The intensity of the 1H envelope of bL12 resonances bound to 14N (15N/13C filtered 1H 1D) is matched by scaling to that of 15N-bound protons (15N/13C edited 1H 1D) in order to quantify the ratio of unlabelled to labelled species. a Intensity profiles of amide resonances in 15N filtered 1H 1D (red) and 15N 1H edited 1D (grey) spectra from a 15N-labelled RNC, in which the background labelling amounts to ~6%. b Intensity profiles of methyl resonances in 13C filtered 1H 1D (red) and 13C edited 1H 1D (grey) spectra from a 13C-labelled RNC. We determine that ~13% of its bL12 resonances are labelled.

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Cassaignau, A., Launay, H., Karyadi, ME. et al. A strategy for co-translational folding studies of ribosome-bound nascent chain complexes using NMR spectroscopy. Nat Protoc 11, 1492–1507 (2016). https://doi.org/10.1038/nprot.2016.101

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