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Title: Spectroscopic studies of labelled proteins in vivo
Author: Haggie, P. M.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1999
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Although metabolic enzymes and metabolic pathways have been well characterised in vitro, understanding of the control of metabolic processes in vivo is relatively poor. The extrapolation of studies performed on purified proteins into the intact cell is questionable. Consequently, there is a need to study enzymes in vivo, however, there are a limited number of techniques available to study specific proteins in a minimally invasive manner. Reported in this thesis are studies in which techniques are developed to permit specific proteins to be distinguished using nuclear magnetic resonance (NMR) and fluorescence visibility. A method to selectively label proteins by the biosynthetic incorporation of amino acid analogs has been developed in Saccharomyces cerevisiae. This method is non-invasive, and the probes introduced into specific proteins are very small and relatively non-perturbing. Using an inducible expression system, pyruvate kinase 1 and citrate synthase 1 were synthesised in vivo and 5-fluorotryptophan in the growth medium was selectively incorporated into the proteins. This labelling permitted the visualisation of these proteins using non-invasive NMR of whole cells. A complementary optical technique was also developed. This method incorporated a fluorescent amino acid analog (5-hydroxytryptophan) into proteins to allow the study of the proteins using more sensitive fluorescence techniques in whole cells. 5-Hydroxytryptophan was successfully incorporated into phosphoglycerate kinase which permitted visualisation of the protein in whole cells using laser scanning confocal microscopy. For both pyruvate kinase 1 and citrate synthase 1, there was evidence of enzyme immobilisation in whole cells. These investigations present direct evidence for the association of enzymes in vivo. The implications of these results in terms of the organisation of metabolism are discussed. The fluorescence studies have important implications both in terms of studying proteins in vivo with microscopy and fluorimetry and in the development of techniques to fluorescently label proteins.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available