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Title: NMR study of the Escherichia coli 70S ribosome particle using residual dipolar couplings
Author: Wang, X.
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2013
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The ribosome is the 2.5-MDa complex that is responsible for production of biologically active polypeptide chains. Crystallographic and cryo-EM studies have revealed the intricate motions of the ribosome during protein synthesis, but less is known about the highly dynamic regions such as L7/L12 stalk protein, which has an important role during this process. Moreover, there is currently very little structural information on the dynamic nascent polypeptide chain (NC), which during its progressive emergence from the ribosome has its first opportunity to acquire structure, folding in a co-translational manner. While the formation of tertiary structure occurs outside of the ribosomal exit tunnel while the NC remains tethered there is little understanding of how this process takes place. NMR spectroscopy, which has the ability to report on both residue-specific structure and dynamics at atomic resolution, has previously been applied to report on mobile regions of the ribosome, such as the L7/L12 stalk protein, and more recently, to the study of co-translational folding of ribosome-nascent chain complexes (RNC). For example, chemical shift studies of RNCs of an immunoglobulin domain (ddFLN-Dom5), have revealed a dynamic RNC capable of adopting the native fold. The motional properties of both L7/L12 and the NC therefore permit the application of NMR strategies, such as residual dipolar couplings (RDC), to derive direct three-dimensional structural information. RDCs encode detailed structural information in the form of distance-independent bond-vector orientations, and can be exploited for both structured and unstructured proteins. This thesis describes steps towards the development of RDC techniques for probing the structural and dynamic properties of RNCs. Initial application to the L7/L12 stalk protein was used as the foundation for subsequent studies of RNCs. Alignment in phage enabled a set of RDCs to be measured for both ribosome-bound and isolated L7/L12, yielding the first direct structural information on the L7/L12 protein in its ribosome-bound form. The application of RDCs to the study of RNCs required extensive development of the strategy for the production of isotopically-labelled RNCs, which resulted in significant improvements in both the yield and quality of the final RNC samples. The RDC study of a model RNC, Dom5+110, highlighted the inherent challenges associated with the finite sample lifetime, low sample concentration and restricted mobility of the RNC. These were addressed with the study of the intrinsically disordered alpha-Syn-RNC, which enabled the measurement of N-H RDCs. A potential interaction between the alpha-Syn NC and the ribosome prevented acquisition of RDCs for all residues, but further studies are underway to improve the quality of the spectra with a view to obtain the complete set of RDC data. Together these data provide a solid foundation for an RDC methodology for application to the ribosome and RNCs, and will enable more detailed structural and dynamical studies of the emerging nascent chain.
Supervisor: Christodoulou, J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available