Characterisation of the prokaryotic elongation factor-Tu with structural mass spectrometry
The elongation factor EF-Tu is an essential and ubiquitous component of prokaryotic mRNA translation. It is directly responsible for the transport of aminoacyl-tRNA to the actively translating ribosome. EF-Tu is a translational GTPase, classically thought to have two conformational extremes induced...
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| Format: | Thesis (University of Nottingham only) |
| Language: | English |
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2025
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| Online Access: | https://eprints.nottingham.ac.uk/80733/ |
| _version_ | 1848801267276578816 |
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| author | Baines, Cameron |
| author_facet | Baines, Cameron |
| author_sort | Baines, Cameron |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | The elongation factor EF-Tu is an essential and ubiquitous component of prokaryotic mRNA translation. It is directly responsible for the transport of aminoacyl-tRNA to the actively translating ribosome. EF-Tu is a translational GTPase, classically thought to have two conformational extremes induced by the binding of either GDP or GTP, the open and closed conformations respectively. It is now hypothesised that, in solution, EF-Tu does not adopt these binary conformations and instead samples a wide range of conformations, with the average becoming more open or closed depending on the bound nucleotide. As an essential prokaryotic protein, EF-Tu presents itself as a desirable antimicrobial target. The elfamycins are a group of bacteriostatic antimicrobials specifically targeting EF-Tu, acting predominantly in one of two ways. Kirromycin-like elfamycins lock EF-Tu in the closed confirmation irrespective of GTP hydrolysis, preventing EF-Tu dissociating from the ribosome. Pulvomycin-like elfamycins simply prevent the binding of tRNA to EF-Tu, acting as a competitive inhibitor.
In this thesis the behaviour of the clinically relevant Acinetobacter baumannii EF-Tu in complex with two contrasting elfamycins, alongside a recently identified prolyl hydroxylase found to target EF-Tu is described. In Chapter 3, the binding of GE2270A, a pulvomycin-like elfamycin, to EF-Tu was explored using native mass spectrometry and carbene footprinting, a covalent protein labelling technique. The binding site of GE2270A was identified, alongside some tentative GE2270A-induced subtle conformational changes. In Chapter 4, the binding of, and global conformational change induced by, the kirromycin-like elfamycin enacyloxin IIa was examined using carbene footprinting. Alongside this, three enacyloxin IIa resistant EF-Tu mutants were produced and characterised using carbene footprinting and native mass spectrometry. In chapter 5, the interactions between an EF-Tu-specific prolyl hydroxylase and EF-Tu were mapped with carbene footprinting. Finally, chapter 6 demonstrated the use of native mass spectrometry to investigate novel enacyloxin IIa analogues and their binding to EF-Tu. Ion mobility spectrometry-mass spectrometry was used to characterise the stability of Escherichia coli EF-Tu in the gas phase. Here, the tertiary structure of EF-Tu was found to be stabilised by ligand binding through collisionally induced unfolding experiments. These results were corroborated with molecular dynamics simulations exploring the thermal stability of EF-Tu in apo and holo states. |
| first_indexed | 2025-11-14T21:04:44Z |
| format | Thesis (University of Nottingham only) |
| id | nottingham-80733 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T21:04:44Z |
| publishDate | 2025 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-807332025-07-30T04:40:12Z https://eprints.nottingham.ac.uk/80733/ Characterisation of the prokaryotic elongation factor-Tu with structural mass spectrometry Baines, Cameron The elongation factor EF-Tu is an essential and ubiquitous component of prokaryotic mRNA translation. It is directly responsible for the transport of aminoacyl-tRNA to the actively translating ribosome. EF-Tu is a translational GTPase, classically thought to have two conformational extremes induced by the binding of either GDP or GTP, the open and closed conformations respectively. It is now hypothesised that, in solution, EF-Tu does not adopt these binary conformations and instead samples a wide range of conformations, with the average becoming more open or closed depending on the bound nucleotide. As an essential prokaryotic protein, EF-Tu presents itself as a desirable antimicrobial target. The elfamycins are a group of bacteriostatic antimicrobials specifically targeting EF-Tu, acting predominantly in one of two ways. Kirromycin-like elfamycins lock EF-Tu in the closed confirmation irrespective of GTP hydrolysis, preventing EF-Tu dissociating from the ribosome. Pulvomycin-like elfamycins simply prevent the binding of tRNA to EF-Tu, acting as a competitive inhibitor. In this thesis the behaviour of the clinically relevant Acinetobacter baumannii EF-Tu in complex with two contrasting elfamycins, alongside a recently identified prolyl hydroxylase found to target EF-Tu is described. In Chapter 3, the binding of GE2270A, a pulvomycin-like elfamycin, to EF-Tu was explored using native mass spectrometry and carbene footprinting, a covalent protein labelling technique. The binding site of GE2270A was identified, alongside some tentative GE2270A-induced subtle conformational changes. In Chapter 4, the binding of, and global conformational change induced by, the kirromycin-like elfamycin enacyloxin IIa was examined using carbene footprinting. Alongside this, three enacyloxin IIa resistant EF-Tu mutants were produced and characterised using carbene footprinting and native mass spectrometry. In chapter 5, the interactions between an EF-Tu-specific prolyl hydroxylase and EF-Tu were mapped with carbene footprinting. Finally, chapter 6 demonstrated the use of native mass spectrometry to investigate novel enacyloxin IIa analogues and their binding to EF-Tu. Ion mobility spectrometry-mass spectrometry was used to characterise the stability of Escherichia coli EF-Tu in the gas phase. Here, the tertiary structure of EF-Tu was found to be stabilised by ligand binding through collisionally induced unfolding experiments. These results were corroborated with molecular dynamics simulations exploring the thermal stability of EF-Tu in apo and holo states. 2025-07-30 Thesis (University of Nottingham only) NonPeerReviewed application/pdf en cc_by https://eprints.nottingham.ac.uk/80733/1/Baines_Cameron_20312252_Corrected.pdf Baines, Cameron (2025) Characterisation of the prokaryotic elongation factor-Tu with structural mass spectrometry. PhD thesis, University of Nottingham. mass spectrometry protein biochemistry structural biology ion mobility native mass spectrometry carbene footprinting EF-Tu translation |
| spellingShingle | mass spectrometry protein biochemistry structural biology ion mobility native mass spectrometry carbene footprinting EF-Tu translation Baines, Cameron Characterisation of the prokaryotic elongation factor-Tu with structural mass spectrometry |
| title | Characterisation of the prokaryotic elongation factor-Tu with structural mass spectrometry |
| title_full | Characterisation of the prokaryotic elongation factor-Tu with structural mass spectrometry |
| title_fullStr | Characterisation of the prokaryotic elongation factor-Tu with structural mass spectrometry |
| title_full_unstemmed | Characterisation of the prokaryotic elongation factor-Tu with structural mass spectrometry |
| title_short | Characterisation of the prokaryotic elongation factor-Tu with structural mass spectrometry |
| title_sort | characterisation of the prokaryotic elongation factor-tu with structural mass spectrometry |
| topic | mass spectrometry protein biochemistry structural biology ion mobility native mass spectrometry carbene footprinting EF-Tu translation |
| url | https://eprints.nottingham.ac.uk/80733/ |