Samuel Mathew

Topic

Effect Of Point Mutations On The Conformation Changes Of PR65 Using Double Nanohole Aperture Tweezer Signals

Department of Electrical and Computer Engineering

Date & location

• Friday, July 5, 2024

• 11:00 A.M.

• Virtual Defence

Reviewers

Supervisory Committee

• Dr. Reuven Gordon, Department of Electrical and Computer Engineering, University of Victoria (Supervisor)

• Dr. Thomas E. Darcie, Department of Electrical and Computer Engineering, UVic (Member)

• Dr. Rustom Bhiladvala, Department of Mechanical Engineering, UVic (Outside Member)

External Examiner

• Dr. Justus Ndukaife, Department of Electrical Engineering, Vanderbilt University 

Chair of Oral Examination

• Dr. Rana El-Sabaawi, Department of Biology, UVic

   

Abstract

A model of the interaction of a Rayleigh particle with a Bethe aperture in terms of the electric dipole of the particle and the effective magnetic dipole of the aperture reveals a polarizability-dependent enhancement in both the trapping potential and the transmission through the Bethe aperture. Since it is known that polarizability is a geometry-dependent material parameter, it is hypothesized that protein conformation changes may be associated with a change in material polarizability. It is further hypothesized that if point mutations, also known as point mutations, impact the conformation of a protein, then they may necessarily alter the material polarizability of a protein too and, through this, the transmission through the aperture of a nano-optical tweezer such as the double nanohole (DNH) aperture tweezer, making it possible to use the optical trapping signals of the DNH aperture tweezer to study the impact of such mutations on a protein such as PR65 – the α-subunit of protein phosphatase 2A (PP2A), which serves as an elastic bridge between the structural and catalytic subunits of PP2A – which has been shown in a previous in silico study to undergo conformation changes in response to mechanical forces such as may be present in an optical tweezer. Thus, DNH apertures were fabricated in a structure of gold-on-glass by colloidal lithography and 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 in terms of parameters including the median transition time between the characteristic jump steps shown by the acquired signals and the root-mean-squared-deviation (RMSD) and corner frequency of the acquired signals. Comparison of these parameters for the mutants with those of PR65 wild type shows that some mutations conferred more stability on PR65 while other iv mutations had a destabilizing effect on PR65. This conclusion is further supported by correlation of these parameters within silico mean contour lengths of PR65 wild type and its six mutants studied. Based on these results, it can be concluded that PR65 undergoes conformation changes that are impacted by substitution mutations. This might have potential for the detection of mutant proteins and for tracking protein mutations and conformational dynamics. The technique developed in the work may also be extended to study protein-ligand binding, although further research to model and characterize conformation changes induced by ligand-binding in the absence of thermo-optical forces would be necessary to extract binding-specific conformation changes from the overall conformation change in this case.