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

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.

journal_name

Nat Protoc

journal_title

Nature protocols

authors

Cassaignau AM,Launay HM,Karyadi ME,Wang X,Waudby CA,Deckert A,Robertson AL,Christodoulou J,Cabrita LD

doi

10.1038/nprot.2016.101

subject

Has Abstract

pub_date

2016-08-01 00:00:00

pages

1492-507

issue

eissn

1754-2189

issn

1750-2799

pii

nprot.2016.101

journal_volume

pub_type

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