Date of Award
August 2024
Degree Type
Thesis
Degree Name
Master of Science
Department
Physics
First Advisor
Ionel V Popa
Abstract
The study of proteins is a well-established field in biophysics with constantly evolving methods for studying different proteins. This thesis explores two ways of studying proteins: using magnetic tweezers and molecular dynamics simulation to learn how proteins unfold and using a novel micro-spectroscopy system with focal correction capabilities to study single-molecule fluorescent proteins.Arrhythmogenic right ventricular cardiomyopathy is a disease that results in the weakening of the heart muscle and has been previously linked to a mutation in the titin protein, the Threonine to Isoleucine missense mutation occurring at residue 2850, or T2850I. One noticeable feature of the mutation T2850I is that it lies directly at the interface between the 10th and the adjacent 11th immunoglobulin domains of human titin (I10 and I11). Using magnetic tweezers experiments to pull on the molecule, my research aimed to answer the question of whether the structure of I10-I11 in tandem is weakened by this mutation. Additionally, using GROMACS, a molecular dynamics simulation software, I pulled on the molecules and monitored different factors such as hydrogen bonds, residue distances, etc. to see what is different between the wild type and the mutated molecules. The second part of my thesis is the applications of a newly developed micro-spectroscopy system for both single molecule and cell measurements. The first application is to measure the photobleaching events of the tetramer and hexamer constructs of the Alexa^488 fluorophore, demonstrating the ability of the instrument to suppress noise and resolve the sample down to the molecular level. The second involves measuring hour-long to day-long fluorescent cell measurements with the bead correction capability. The second application involves the study of the cellular actin dynamics by looking at the filopodial activities of the human embryonic kidney (HEK) 293 cells with filopodia tagged with the green fluorescent protein (meGFP). HEK 293 cells whose actin filaments are tagged with meGFP are then fixed and visualized. The resulting images of the fixed cells are analyzed using the stochastic optical reconstruction microscopy method to make a super-resolution image of the cytoskeleton of the cell.
Recommended Citation
Phan, Binh Thai, "PROBING FLUORESCENT AND MECHANICAL PROPERTIES OF BIOMOLECULES USING SINGLE MOLECULE TECHNIQUES" (2024). Theses and Dissertations. 3614.
https://dc.uwm.edu/etd/3614