Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.790856
Title: Development of novel approaches towards the study of ribosome bound nascent chain complexes by NMR spectroscopy
Author: Paton, J. F. S.
ISNI:       0000 0004 8499 7716
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2017
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Abstract:
Co-translational folding is a fundamental process in biology studied primarily using biochemical and biophysical techniques. The dynamic nascent chain has largely eluded high resolution structural study, although NMR spectroscopy is recently emerging as one of the principal techniques for acquiring a dynamic and structural understanding of the nascent chain due its advantages in investigating dynamic systems. As such, NMR has lead to a rich understanding of ribosome-bound nascent chains (RNCs) in terms of their dynamics and folding on the ribosome. The aim of this project is to explore the NMR observable properties that can be manipulated according to the kind of system one chooses to study. In particular, the exploitation of the fast exchange regime in NMR spectroscopy has not been applied to the study of RNCs. This is despite the potential advantages fast exchange can show over slow exchange for samples of low concentration and stability, which make it potentially very advantageous for the study of RNCs. We briefly describe the previous approaches that have been successful in producing nascent chain samples that can be studied by NMR, namely -synuclein, an intrinsically disordered protein (IDP), that allows for investigations of interactions with the ribosome surface, and domain 5 of the gelation factor of Dictyostelium discoideum, where co-translational folding has been probed by the development of constructs that possess varying degrees of structure whilst attached to the ribosome. Following this, an argument is presented for the advantages in exploiting the fast exchange regime, as the domain 5 system is in slow exchange on the NMR timescale, which leads to certain limitations. This is followed by the design and development of translation stalled nascent chains of fast folding domains. We present NMR characterisation of the folding of two such fast folding proteins, the GA module and the HP36 villin headpiece domain. This is followed by NMR investigations into a translation stalled construct of the GA module, a small albumin binding domain. Further to this, we also investigate the interaction between -synuclein and the ribosome surface by measurement of amide proton relaxation rates of -synuclein over a titration of varying -syn concentrations. Finally, we show the results of attempts to incorporate 3-fluorotyrosine into proteins and RNC samples, and show how this can be used as a unique NMR probe. We show that this approach allows one to acquire NMR spectra of RNCs with no background signal from the ribosome, and that the sensitivity of the fluorine chemical shift to its environment therefore has the potential to uncover rich information about the ribosome surface and the effect it has on interacting species.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.790856  DOI: Not available
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