Effect of point mutations on the conformation changes of PR65 using double nanohole aperture tweezer signals
| dc.contributor.author | Mathew, Samuel | |
| dc.contributor.supervisor | Gordon, Reuven | |
| dc.date.accessioned | 2024-07-12T17:47:24Z | |
| dc.date.available | 2024-07-12T17:47:24Z | |
| dc.date.issued | 2024 | |
| dc.degree.department | Department of Electrical and Computer Engineering | |
| dc.degree.level | Doctor of Philosophy PhD | |
| dc.description.abstract | The purpose of this research was to investigate the effect of point mutations on the conformation changes of PR65 using double nanohole (DNH) optical tweezers. It explored the following questions: how does a dielectric nanoparticle interact with a nanoaperture? Could the way a nanoparticle interacts with a nanoaperture be exploited for probing protein conformation changes using aperture tweezers such as the DNH optical tweezer? What can be said of the behavior of PR65 when trapped in a DNH optical tweezer? Could the behavior of PR65 when trapped in a DNH optical tweezer be explained in terms of how PR65 interacts with the DNH aperture? Does the behavior of PR65 when trapped in a DNH change with point mutation? How might this be exploited for probing the impact of point mutations on the conformational dynamics of PR65 using DNH optical tweezers? The study has implications for tracking mutations in proteins as well as for drug discovery and testing. Methods employed included both theoretical modelling and experimental measurements using the DNH optical tweezer. We modelled the interaction between a nanoaperture and a dielectric nanoparticle in terms of a simple dipole-dipole interaction based on the Rayleigh scattering and Bethe aperture theorems. Our model showed that the interaction enhanced both the trapping potential and the transmission through the aperture in accordance with the self-induced back-action (SIBA) effect, in which a nanoparticle interacting with a focused laser beam aids in its own trapping. The model agreed quite well with numerical simulations performed in Lumerical and revealed that the motion of a particle trapped in an optical tweezer can be used to probe changes in the shape and size of a particle. This is because changes in the shape and size of a particle in an optical tweezer will alter the polarizability of such a particle, and therefore the restoring force that it felt in the tweezer. This will manifest as differences in root-mean-squared displacement (RMSD) and corner frequency characteristic of the motion of the particle in the optical tweezer between one conformation and another. We thus formulated a hypothesis that if conformation changes induced by point mutations alter the material polarizability of PR65, then the DNH optical tweezer signals acquired by trapping each mutant PR65 will have different RMSDs and corner frequencies from that of wild type PR65. To test this hypothesis, DNH optical tweezers fabricated by colloidal lithography were used to trap PR65 wild type and six of its mutants at a laser power of ~ 22 mW. The resulting optical signals were captured using an avalanche photodiode (APD) connected to a digital USB-4771A data acquisition module and analyzed using MATLAB. Parameters extracted from the acquired signals and studied included the median transition time between the characteristic jump states shown by the acquired signals and the RMSD and corner frequency of the acquired signals. These parameters were higher for some of the mutants of PR65 and lower for others in comparison with wild type PR65. Correlation of the RMSDs with in silico mean contour lengths of PR65 wild type and six of its mutants studied was also consistent with this conclusion, and in agreement with our hypothesis. These results imply that PR65 undergoes conformation changes that are impacted by substitution mutations, with some mutations causing PR65 to assume an elongated conformation and other mutations causing PR65 to assume a more compact conformation. | |
| dc.description.scholarlevel | Graduate | |
| dc.identifier.bibliographicCitation | Mathew, S., & Gordon, R. (2023). Self-induced back-action for aperture trapping: Bethe-Rayleigh theory. Optics Express, 31(26), 44190–44198. https://doi.org/10.1364/OE.510635 | |
| dc.identifier.bibliographicCitation | Banerjee, A., Mathew, S., Naqvi, M. M., Yilmaz, S. Z., Zacharopoulou, M., Doruker, P., Kumita, J. R., Yang, S.-H., Gur, M., Itzhaki, L. S., Gordon, R., & Bahar, I. (2024). Influence of point mutations on PR65 conformational adaptability: Insights from molecular simulations and nanoaperture optical tweezers. Science Advances, 10(22), eadn2208-. https://doi.org/10.1126/sciadv.adn2208 | |
| dc.identifier.bibliographicCitation | Ravindranath, A. L., Shariatdoust, M. S., Mathew, S., & Gordon, R. (2019). Colloidal lithography double-nanohole optical trapping of nanoparticles and proteins. Optics Express, 27(11), 16184–16194. https://doi.org/10.1364/OE.27.016184 | |
| dc.identifier.uri | https://hdl.handle.net/1828/16747 | |
| dc.language | English | eng |
| dc.language.iso | en | |
| dc.rights | Available to the World Wide Web | |
| dc.subject | Plasmonics | |
| dc.subject | Double nanohole (DNH) | |
| dc.subject | Colloidal lithography | |
| dc.subject | Point mutations | |
| dc.subject | Optical trapping | |
| dc.subject | Allostery | |
| dc.subject | Self-induced back-action (SIBA) effect | |
| dc.subject | PR65 | |
| dc.subject | Protein phosphatase 2A (PP2A) | |
| dc.subject | Power spectral density | |
| dc.subject | Corner frequency | |
| dc.subject | Root-mean-squared displacement (RMSD) | |
| dc.subject | Protein structure | |
| dc.subject | Optical tweezers | |
| dc.title | Effect of point mutations on the conformation changes of PR65 using double nanohole aperture tweezer signals | |
| dc.type | Thesis |