Enzyme mechanisms studied by single molecule optical sensing
Lead Research Organisation:
University of Exeter
Department Name: Physics
Abstract
The project will involve a combination of cutting edge physical and biochemical methods. Ubiquitination reactions will be studied using plasmonically enhanced whispering gallery mode (WGM) microcavity sensing. This is the first optical technique capable of directly monitoring structural changes within individual biomolecules such as proteins. A major aim will be establishing whether each type of ubiquitin linkage has a unique WGM signature. Biochemical and chemical biology experiments with Drs Bagby and Whitley at Bath will include ubiquitination assays, and production of ubiquitin and enzymes (ligases and deubiquitinases) modified for immobilisation on gold nanoparticles that are used in WGM sensing. Single molecule WGM sensing studies of ubiquitin chain assembly will be conducted at Exeter with Professor Vollmer's group.
Ubiquitination is an important post-translational modification involved in modulation and regulation of protein function in many processes in most, if not all, eukaryotic cell types; ubiquitination goes awry in numerous diseases. Ubiquitination involves the covalent attachment to a target protein (the "substrate") of one molecule, or multiple molecules in chains, of a small protein called ubiquitin. As described below, different types of ubiquitin chains can be assembled; this is important because chain type determines the biological outcome of ubiquitination (e.g. whether the substrate protein is degraded, or its function or location is affected), but currently it is difficult to determine chain type. The aim is to develop a rapid, accurate and user-friendly method to identify the type of ubiquitin chain assembled by any E3 ubiquitin ligase, at the same time providing new insights into ubiquitination mechanism and kinetics.
Ubiquitination, catalysed by ubiquitin ligases, involves covalent conjugation of ubiquitin to the protein substrate via formation of an isopeptide bond between ubiquitin's C-terminal carboxylate and the substrate's N-terminal amine or -amino group of a lysine residue. In many cases a chain of ubiquitin molecules is assembled on a substrate protein whereby a ubiquitin monomer is linked to the chain via any one of seven lysines or N-terminus. Ubiquitin chains can involve a single type of lysine linkage or mixed linkages, with each linkage producing a different degree of flexibility and repertoire of conformational states. Since chain type determines biological outcome, this project will provide detailed new insights into the relationships between E3 ligase function and resulting phenotype, and will therefore potentially advance understanding of the relationships between particular ubiquitin ligases and health and disease.
Ubiquitination is an important post-translational modification involved in modulation and regulation of protein function in many processes in most, if not all, eukaryotic cell types; ubiquitination goes awry in numerous diseases. Ubiquitination involves the covalent attachment to a target protein (the "substrate") of one molecule, or multiple molecules in chains, of a small protein called ubiquitin. As described below, different types of ubiquitin chains can be assembled; this is important because chain type determines the biological outcome of ubiquitination (e.g. whether the substrate protein is degraded, or its function or location is affected), but currently it is difficult to determine chain type. The aim is to develop a rapid, accurate and user-friendly method to identify the type of ubiquitin chain assembled by any E3 ubiquitin ligase, at the same time providing new insights into ubiquitination mechanism and kinetics.
Ubiquitination, catalysed by ubiquitin ligases, involves covalent conjugation of ubiquitin to the protein substrate via formation of an isopeptide bond between ubiquitin's C-terminal carboxylate and the substrate's N-terminal amine or -amino group of a lysine residue. In many cases a chain of ubiquitin molecules is assembled on a substrate protein whereby a ubiquitin monomer is linked to the chain via any one of seven lysines or N-terminus. Ubiquitin chains can involve a single type of lysine linkage or mixed linkages, with each linkage producing a different degree of flexibility and repertoire of conformational states. Since chain type determines biological outcome, this project will provide detailed new insights into the relationships between E3 ligase function and resulting phenotype, and will therefore potentially advance understanding of the relationships between particular ubiquitin ligases and health and disease.
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/T008741/1 | 01/10/2020 | 30/09/2028 | |||
| 2401633 | Studentship | BB/T008741/1 | 01/10/2020 | 30/09/2024 | Matthew Houghton |
| Description | - A new method of understanding how molecules absorb different types of light, and how this can be applied to creating new analysis methods for biomolecules. - Discovered a new method for exerting forces on enzymes using light, which could allow us to control enzymes for biotechnology use. |
| Exploitation Route | - Understanding interactions of molecules and light in this way could provide additional information as to how this optoplamonic tehcnique works and accesses information about molecules under test. - Controlling enzymes with light could allow a platform to be made that won't damage the enzyme or require extensive enzyme preparation/modification, reuslting in a lower-cost and more effective technique than established methods. |
| Sectors | Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Description | Molecular Mechanics of Enzymes |
| Amount | £2,086,999 (GBP) |
| Funding ID | EP/T002875/1 |
| Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 04/2019 |
| End | 09/2022 |
| Title | Thermo-Optoplasmonic Sensing |
| Description | Thermo-Optoplasmonic (TOP) sensing is a new method for detecting absorption of light by molecules and estimating their absorption cross-sections. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2022 |
| Provided To Others? | Yes |
| Impact | - Publication |
| URL | https://doi.org/10.1364/SENSORS.2022.SW3E.6 |
| Description | Enzyme Thermodynamics and Forces |
| Organisation | University of Exeter |
| Department | School of Physics |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Mr Houghton has collected data, analysed results, prepared samples and written the publication as the primary author. |
| Collaborator Contribution | Dr Rubio was responsible for theoretical work and analysis. Dr Toropov was responsible for data collection and analysis. |
| Impact | - Publication soon |
| Start Year | 2022 |
| Description | Enzyme Thermodynamics and Forces |
| Organisation | University of Southampton |
| Department | Optoelectronics Research Centre |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Mr Houghton has collected data, analysed results, prepared samples and written the publication as the primary author. |
| Collaborator Contribution | Dr Rubio was responsible for theoretical work and analysis. Dr Toropov was responsible for data collection and analysis. |
| Impact | - Publication soon |
| Start Year | 2022 |
| Description | Thermo-Optoplasmonic Sensing |
| Organisation | National Time Service Center |
| Country | China |
| Sector | Public |
| PI Contribution | Collaboration with Nikita Toropov, of the Optoelectronics Institute at the University of Southampton, and Deshui Yu, of National Time Service Center, Chinese Academy of Sciences, in writing a manuscript for publication, continuing work that Dr Toropov and Mr Houghton worked on together at the University of Exeter. Mr Houghton is co-primary author- experimental design, aquisition, writing and analysis. |
| Collaborator Contribution | Dr Toropov is the primary author on this paper. They were involved in data aquisition and analysis when at University of exeter, but now involved in writing and analysis from University of Southampton. Dr Yu is a secondary author and involved in data analysis and writing of the publication, carrying on work started when also at the University of Exeter. |
| Impact | - Publication soon |
| Start Year | 2023 |
| Description | Thermo-Optoplasmonic Sensing |
| Organisation | University of Southampton |
| Department | Optoelectronics Research Centre |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Collaboration with Nikita Toropov, of the Optoelectronics Institute at the University of Southampton, and Deshui Yu, of National Time Service Center, Chinese Academy of Sciences, in writing a manuscript for publication, continuing work that Dr Toropov and Mr Houghton worked on together at the University of Exeter. Mr Houghton is co-primary author- experimental design, aquisition, writing and analysis. |
| Collaborator Contribution | Dr Toropov is the primary author on this paper. They were involved in data aquisition and analysis when at University of exeter, but now involved in writing and analysis from University of Southampton. Dr Yu is a secondary author and involved in data analysis and writing of the publication, carrying on work started when also at the University of Exeter. |
| Impact | - Publication soon |
| Start Year | 2023 |
| Description | Opening of Faculty of Life sciences (Poster Presentation) |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | - Poster on research was presented at the opening of the Faculty of Life Sciences at the University of Exeter. Questions were asked on the research and it's place in the department. |
| Year(s) Of Engagement Activity | 2022 |