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Yousefi, A, 2024. Optical nanotweezers for characterising unmodified proteins at the single-molecule level. PhD, Nottingham Trent University.
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Abstract
Proteins, essential macromolecules found in all living organisms, are responsible for various biological functions, including enzymatic activity, structural support, and regulation of cellular processes. Understanding their mechanisms such as protein folding, and protein-protein interaction is crucial for revealing cellular functions and disease pathways. Single-molecule characterisation techniques offer detailed insights into protein behaviour, such as conformational changes and dynamic interactions, which were previously difficult to study using bulk methods. However, conventional single-molecule techniques, such as single-molecule Förster resonance energy transfer (smFRET) and cryogenic electron microscopy (cryo-EM), have notable limitations. smFRET necessitates chemical modifications, which hinder the ability to monitor rapid protein dynamics in their native state. On the other hand, cryo-EM is inherently non-continuous, capturing only static snapshots of protein movements. As a result, these methods mainly lack utility for studying the single-molecule kinetic reactions of proteins.
This thesis employs optical nanotweezers using plasmonic double-nanohole (DNH) structures to trap individual, unlabelled proteins, through driving the oscillation of free-electrons, known as surface plasmons, excited by light. The light passing through the DNHs focuses the electric field into nanometre-scale regions known as hotspots, which are sensitive to refractive index changes in these areas. Since protein structural changes are linked to variations in the refractive index, this approach enables us to observe conformational changes in trapped proteins with high sensitivity. We applied this approach to study ferritin, a ubiquitous protein present in nearly all living organisms. Ferritin plays a critical role in iron storage and regulation, which is essential for maintaining iron homeostasis and supporting overall physiological function.
This thesis will describe trapping and monitoring the structural dynamics of apoferritin (protein without iron core) and holo-ferritin (protein with iron core) in real-time. Notably, we monitored the iron-loading process in a single apo-ferritin as it converted to a holo-ferritin in situ. Furthermore, the linear relationship between light transmission through the DNH and the size of the trapped particles enabled us to track the disassembly of single ferritin molecules in acidic environments. Our results indicated that the protein disassembly occurs in a stepwise manner, with subunits disassembling cooperatively, emphasising the important role of intermediate subunits in the process.
This thesis demonstrates the benefits of, optical nanotweezers, as a label-free approach, to study individual proteins in their native environment without modification and in real time. Our results enhance the understanding of ferritin, particularly its disassembly process and the kinetics of iron release, which could aid in the design of drug delivery carriers and the development of medical treatments and pharmaceuticals. Moreover, the versatile nature of optical nanotweezers allows for the extension of this approach to investigate a wide range of other proteins, providing a valuable understanding of their structure, dynamics, and interactions, and further broadening the potential applications in biomedical research.
| Item Type: | Thesis |
|---|---|
| Creators: | Yousefi, A. |
| Contributors: | Name Role NTU ID ORCID |
| Date: | September 2024 |
| Rights: | The copyright in this work is held by the author. You may copy up to 5% of this work for private study, or personal, non-commercial research. Any re-use of the information contained within this document should be fully referenced, quoting the author, title, university, degree level and pagination. Queries or requests for any other use, or if a more substantial copy is required, should be directed to the author. |
| Divisions: | Schools > School of Science and Technology |
| Record created by: | Laura Borcherds |
| Date Added: | 10 Jun 2025 09:24 |
| Last Modified: | 10 Jun 2025 09:24 |
| URI: | https://irep.ntu.ac.uk/id/eprint/53714 |
https://orcid.org/0000-0001-9268-4793Actions (login required)
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