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

Cassaignau, AM; Launay, HM; Karyadi, ME; Wang, X; Waudby, CA; Deckert, A; Robertson, AL; ... Cabrita, LD; + view all (2016) A strategy for co-translational folding studies of ribosome-bound nascent chain complexes using NMR spectroscopy. Nature Protocols , 11 (8) pp. 1492-1507. 10.1038/nprot.2016.101. Green open access

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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.

Type: Article
Title: A strategy for co-translational folding studies of ribosome-bound nascent chain complexes using NMR spectroscopy
Location: England
Open access status: An open access version is available from UCL Discovery
DOI: 10.1038/nprot.2016.101
Publisher version: http://doi.org/10.1038/nprot.2016.101
Language: English
Additional information: © 2016 Nature America, Inc. All rights reserved.
Keywords: Biopolymers in vivo; Protein folding; Ribosome; Solution-state NMR
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology
URI: http://discovery.ucl.ac.uk/id/eprint/1506184
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