Journal
NATURE PROTOCOLS
Volume 11, Issue 8, Pages 1492-1507Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2016.101
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Funding
- New Investigator Award [BBSRC BBG0156511]
- Wellcome Trust Investigator Award [097806/Z/11/Z]
- AlphaOne Foundation
- Medical Research Council [MC_U117533887] Funding Source: researchfish
- The Francis Crick Institute [10029] Funding Source: researchfish
- MRC [MC_U117533887] Funding Source: UKRI
- Wellcome Trust [097806/Z/11/Z] Funding Source: Wellcome Trust
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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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