Related ArticlesA strategy for co-translational folding studies of ribosome-bound nascent chain complexes using NMR spectroscopy.
Nat Protoc. 2016 Aug;11(8):1492-1507
Authors: Cassaignau AM, Launay HM, Karyadi ME, Wang X, Waudby CA, Deckert A, Robertson AL, Christodoulou J, Cabrita LD
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.
PMID: 27466710 [PubMed - as supplied by publisher]
[NMR paper] Increasing the sensitivity of NMR diffusion measurements by paramagnetic longitudinal relaxation enhancement, with application to ribosome-nascent chain complexes.
Increasing the sensitivity of NMR diffusion measurements by paramagnetic longitudinal relaxation enhancement, with application to ribosome-nascent chain complexes.
Related Articles Increasing the sensitivity of NMR diffusion measurements by paramagnetic longitudinal relaxation enhancement, with application to ribosome-nascent chain complexes.
J Biomol NMR. 2015 Aug 8;
Authors: Chan SH, Waudby CA, Cassaignau AM, Cabrita LD, Christodoulou J
Abstract
The translational diffusion of macromolecules can be examined non-invasively by...
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Increasing the sensitivity of NMR diffusion measurements by paramagnetic longitudinal relaxation enhancement, with application to ribosomeâ??nascent chain complexes
Increasing the sensitivity of NMR diffusion measurements by paramagnetic longitudinal relaxation enhancement, with application to ribosomeâ??nascent chain complexes
Abstract
The translational diffusion of macromolecules can be examined non-invasively by stimulated echo (STE) NMR experiments to accurately determine their molecular sizes. These measurements can be important probes of intermolecular interactions and protein folding and unfolding, and are crucial in monitoring the integrity of large macromolecular assemblies...
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[NMR paper] Protein folding on the ribosome studied using NMR spectroscopy.
Protein folding on the ribosome studied using NMR spectroscopy.
Protein folding on the ribosome studied using NMR spectroscopy.
Prog Nucl Magn Reson Spectrosc. 2013 Oct;74C:57-75
Authors: Waudby CA, Launay H, Cabrita LD, Christodoulou J
Abstract
NMR spectroscopy is a powerful tool for the investigation of protein folding and misfolding, providing a characterization of molecular structure, dynamics and exchange processes, across a very wide range of timescales and with near atomic resolution. In recent years NMR methods have also been...
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[NMR paper] Backbone and side chain NMR assignments for the ribosome assembly factor Nop6 from Saccharomyces cerevisiae.
Backbone and side chain NMR assignments for the ribosome assembly factor Nop6 from Saccharomyces cerevisiae.
Backbone and side chain NMR assignments for the ribosome assembly factor Nop6 from Saccharomyces cerevisiae.
Biomol NMR Assign. 2013 Aug 7;
Authors: Wurm JP, Lioutikov A, Kötter P, Entian KD, Wöhnert J
Abstract
The Saccharomyces cerevisiae Nop6 protein is involved in the maturation of the small ribosomal subunit. It contains a central RNA binding domain and a predicted C-terminal coiled-coil domain. Here we report the almost...
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Protein folding on the ribosome studied using NMR spectroscopy
Protein folding on the ribosome studied using NMR spectroscopy
Publication date: Available online 27 July 2013
Source:Progress in Nuclear Magnetic Resonance Spectroscopy</br>
Author(s): Christopher A. Waudby , Hélène Launay , Lisa D. Cabrita , John Christodoulou</br>
NMR spectroscopy is a powerful tool for the investigation of protein folding and misfolding, providing a characterization of molecular structure, dynamics and exchange processes, across a very wide range of timescales and with near atomic resolution. In recent years NMR methods have also been...
Backbone and side chain NMR resonance assignments for an archaeal homolog of the endonuclease Nob1 involved in ribosome biogenesis.
Backbone and side chain NMR resonance assignments for an archaeal homolog of the endonuclease Nob1 involved in ribosome biogenesis.
Backbone and side chain NMR resonance assignments for an archaeal homolog of the endonuclease Nob1 involved in ribosome biogenesis.
Biomol NMR Assign. 2011 Jul 6;
Authors: Veith T, Wurm JP, Duchardt-Ferner E, Weis B, Martin R, Safferthal C, Bohnsack MT, Schleiff E, Wöhnert J
Eukaryotic ribosome biogenesis requires the concerted action of ~200 auxiliary protein factors on the nascent ribosome. For many of these...
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[NMR paper] Heteronuclear NMR studies of the specificity of the post-translational modification o
Heteronuclear NMR studies of the specificity of the post-translational modification of biotinyl domains by biotinyl protein ligase.
Related Articles Heteronuclear NMR studies of the specificity of the post-translational modification of biotinyl domains by biotinyl protein ligase.
FEBS Lett. 2000 Aug 18;479(3):93-8
Authors: Reche PA, Howard MJ, Broadhurst RW, Perham RN
The lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes and the biotinyl domains of biotin-dependent enzymes have homologous structures, but the target lysine...
A strategy for co-translational folding studies of ribosome-bound nascent chain complexes using NMR spectroscopy.
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