Development of novel molecular probes to study cellular behaviour using NMR: i) Development of a novel 19F NMR probe to monitor the dynamic self-assembly of high molecular weight proteins in cells ii) Mechanistic analysis of lipoamide as a potential ALS drug by stable isotope labelling and quantitative NMR
<p>This DPhil thesis describes the novel analytical methodologies developed by the author using biomolecular Nuclear Magnetic Resonance (NMR) spectroscopy. These new bio-NMR methods address the current limitations in modern NMR spectroscopy and can be used to study the structure and dynamics o...
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Thesis |
| Language: | English |
| Published: |
2023
|
| Subjects: |
| _version_ | 1811139828166688768 |
|---|---|
| author | Kishigami, S |
| author2 | Casablancas Antras, V |
| author_facet | Casablancas Antras, V Kishigami, S |
| author_sort | Kishigami, S |
| collection | OXFORD |
| description | <p>This DPhil thesis describes the novel analytical methodologies developed by the author using biomolecular Nuclear Magnetic Resonance (NMR) spectroscopy. These new bio-NMR methods address the current limitations in modern NMR spectroscopy and can be used to study the structure and dynamics of target biomolecules in complex cellular environments. More specifically, the author synthesised small molecules labelled with stable isotopes which are NMR active and can be used for molecular biology experiments to gain useful biological insights. Chapter 1 clarifies the strengths and weaknesses of the modern NMR methodologies and describes the research backgrounds of the DPhil research projects. Then, Chapter 2 describes a <sup>15</sup>N isotope labelling technique and a heteronuclear NMR method to detect the cellular uptake of a potent drug candidate by the mammalian cells. In Chapter 3, the motivation for the development of a novel (<sup>13</sup>CF<sub>3</sub>) NMR probe is explained with the synthetic procedures to prepare the molecules labelled with the (<sup>13</sup>CF<sub>3</sub>) moieties. Chapter 4 provides a detailed explanation of a molecular biology methodology to deliver the unnatural (<sup>13</sup>CF<sub>3</sub>) label to proteins of interest. The NMR results obtained from the (<sup>13</sup>CF<sub>3</sub>)-labelled Hsp 16.5 protein sample in Chapter 5 confirmed a significant potential of the (<sup>13</sup>CF<sub>3</sub>)-group to study structures and dynamics of high molecular weight biomacromolecules. A unique application of the fluorine NMR probe to label spider silk protein fibre is introduced in Chapter 6. The conclusions and outlook of the work described in this DPhil thesis are presented in Chapter 7. Overall, this DPhil thesis aims to provide some unique NMR methodologies for studying the behaviour of biomolecules in complex cellular contexts and contribute to the understanding of biological phenomena by using these NMR techniques.</p> |
| first_indexed | 2024-09-25T04:12:17Z |
| format | Thesis |
| id | oxford-uuid:7e0d4be0-8620-4124-bdfd-330d9c9b02be |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-09-25T04:12:17Z |
| publishDate | 2023 |
| record_format | dspace |
| spelling | oxford-uuid:7e0d4be0-8620-4124-bdfd-330d9c9b02be2024-06-27T13:21:59ZDevelopment of novel molecular probes to study cellular behaviour using NMR: i) Development of a novel 19F NMR probe to monitor the dynamic self-assembly of high molecular weight proteins in cells ii) Mechanistic analysis of lipoamide as a potential ALS drug by stable isotope labelling and quantitative NMRThesishttp://purl.org/coar/resource_type/c_db06uuid:7e0d4be0-8620-4124-bdfd-330d9c9b02beProtein complexesPulsed nuclear magnetic resonanceStable isotope tracersOrganofluorine compoundsBiochemistryBiophysicsOrganic chemistryEnglishHyrax Deposit2023Kishigami, SCasablancas Antras, VVincent, KBaldwin, AJDavis, BGEaton, JFierz, B<p>This DPhil thesis describes the novel analytical methodologies developed by the author using biomolecular Nuclear Magnetic Resonance (NMR) spectroscopy. These new bio-NMR methods address the current limitations in modern NMR spectroscopy and can be used to study the structure and dynamics of target biomolecules in complex cellular environments. More specifically, the author synthesised small molecules labelled with stable isotopes which are NMR active and can be used for molecular biology experiments to gain useful biological insights. Chapter 1 clarifies the strengths and weaknesses of the modern NMR methodologies and describes the research backgrounds of the DPhil research projects. Then, Chapter 2 describes a <sup>15</sup>N isotope labelling technique and a heteronuclear NMR method to detect the cellular uptake of a potent drug candidate by the mammalian cells. In Chapter 3, the motivation for the development of a novel (<sup>13</sup>CF<sub>3</sub>) NMR probe is explained with the synthetic procedures to prepare the molecules labelled with the (<sup>13</sup>CF<sub>3</sub>) moieties. Chapter 4 provides a detailed explanation of a molecular biology methodology to deliver the unnatural (<sup>13</sup>CF<sub>3</sub>) label to proteins of interest. The NMR results obtained from the (<sup>13</sup>CF<sub>3</sub>)-labelled Hsp 16.5 protein sample in Chapter 5 confirmed a significant potential of the (<sup>13</sup>CF<sub>3</sub>)-group to study structures and dynamics of high molecular weight biomacromolecules. A unique application of the fluorine NMR probe to label spider silk protein fibre is introduced in Chapter 6. The conclusions and outlook of the work described in this DPhil thesis are presented in Chapter 7. Overall, this DPhil thesis aims to provide some unique NMR methodologies for studying the behaviour of biomolecules in complex cellular contexts and contribute to the understanding of biological phenomena by using these NMR techniques.</p> |
| spellingShingle | Protein complexes Pulsed nuclear magnetic resonance Stable isotope tracers Organofluorine compounds Biochemistry Biophysics Organic chemistry Kishigami, S Development of novel molecular probes to study cellular behaviour using NMR: i) Development of a novel 19F NMR probe to monitor the dynamic self-assembly of high molecular weight proteins in cells ii) Mechanistic analysis of lipoamide as a potential ALS drug by stable isotope labelling and quantitative NMR |
| title | Development of novel molecular probes to study cellular behaviour using NMR:
i) Development of a novel 19F NMR probe to monitor the dynamic self-assembly of high molecular weight proteins in cells
ii) Mechanistic analysis of lipoamide as a potential ALS drug by stable isotope labelling and quantitative NMR |
| title_full | Development of novel molecular probes to study cellular behaviour using NMR:
i) Development of a novel 19F NMR probe to monitor the dynamic self-assembly of high molecular weight proteins in cells
ii) Mechanistic analysis of lipoamide as a potential ALS drug by stable isotope labelling and quantitative NMR |
| title_fullStr | Development of novel molecular probes to study cellular behaviour using NMR:
i) Development of a novel 19F NMR probe to monitor the dynamic self-assembly of high molecular weight proteins in cells
ii) Mechanistic analysis of lipoamide as a potential ALS drug by stable isotope labelling and quantitative NMR |
| title_full_unstemmed | Development of novel molecular probes to study cellular behaviour using NMR:
i) Development of a novel 19F NMR probe to monitor the dynamic self-assembly of high molecular weight proteins in cells
ii) Mechanistic analysis of lipoamide as a potential ALS drug by stable isotope labelling and quantitative NMR |
| title_short | Development of novel molecular probes to study cellular behaviour using NMR:
i) Development of a novel 19F NMR probe to monitor the dynamic self-assembly of high molecular weight proteins in cells
ii) Mechanistic analysis of lipoamide as a potential ALS drug by stable isotope labelling and quantitative NMR |
| title_sort | development of novel molecular probes to study cellular behaviour using nmr i development of a novel 19f nmr probe to monitor the dynamic self assembly of high molecular weight proteins in cells ii mechanistic analysis of lipoamide as a potential als drug by stable isotope labelling and quantitative nmr |
| topic | Protein complexes Pulsed nuclear magnetic resonance Stable isotope tracers Organofluorine compounds Biochemistry Biophysics Organic chemistry |
| work_keys_str_mv | AT kishigamis developmentofnovelmolecularprobestostudycellularbehaviourusingnmridevelopmentofanovel19fnmrprobetomonitorthedynamicselfassemblyofhighmolecularweightproteinsincellsiimechanisticanalysisoflipoamideasapotentialalsdrugbystableisotopelabellingandquantitativenm |