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In vitro and in-cell NMR approaches to the study of ribosome-nascent chain complexes

Chan, SHS; (2017) In vitro and in-cell NMR approaches to the study of ribosome-nascent chain complexes. Doctoral thesis , UCL (University College London).

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Abstract

The ubiquitous ribosome is responsible for the synthesis of proteins, which generally require correct three-dimensional folds for their biologically active function. As the nascent polypeptide progressively emerges from the ribosomal exit tunnel, it is able to acquire structure concurrently with its biosynthesis while it is exposed to the densely packed and crowded cellular environment. NMR spectroscopy is uniquely able to probe such dynamic structures and folding processes at near-atomic resolution, and its application to translationally stalled ribosome-nascent chain complexes (RNCs) has allowed us to begin to understand co-translational protein folding in the cell. However, these studies are frequently restricted by weak signals arising from high molecular weight of ~2.4 MDa, the resulting low maximum achievable concentrations, and limited sample lifetimes of typically just a few hours, which present a major barrier to progress in this field. To overcome the substantial spectroscopic challenges of NMR studies of RNCs, we have employed the use of the paramagnetic longitudinal relaxation enhancement (PLRE) agent NiDO2A in combination with longitudinal relaxation-optimised experiments, and report improvements of up to 4.8-fold in the sensitivities of two-dimensional 1H-15N NMR correlation spectra and NMR diffusion measurements that are crucial in assessing the integrity of unstable RNCs. We describe how the PLRE approach can be exploited in more complex NMR experiments to extract direct structural information on RNCs of an immunoglobulin-like domain using three-dimensional NMR and residual dipolar coupling measurements, which have previously been unattainable due to low spectroscopic sensitivity. We further describe a strategy to translate current in vitro RNC studies into NMR investigations within living cells, and address how ribosome-bound species can be distinguished from those isolated from the ribosome in vivo. Experiments in reconstituted cytosol revealed that macromolecular crowding effects perturb the structure and dynamics of FLN5. We anticipate our studies will significantly advance the application of NMR to large macromolecular assemblies, and to studies of protein folding under increasingly more physiologically relevant conditions.

Type: Thesis (Doctoral)
Title: In vitro and in-cell NMR approaches to the study of ribosome-nascent chain complexes
Event: UCL (University College London)
Language: English
UCL classification: UCL > School of Life and Medical Sciences
UCL > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Structural and Molecular Biology
URI: http://discovery.ucl.ac.uk/id/eprint/1553360
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