Development of optical trapping assays to investigate bacteriophage T4 packaging

Viruses, as the most abundant biological entity on earth, have small size but carry out multiple fundamental biological functions, including genome packaging. Understanding how the packaging motors work not only extends our knowledge of fundamental biology but also benefits the development of drug delivery. Bacteriophage T4, a model system for virus genome packaging, has been widely studied in basic structures and functions. Its multi-subunit ring-shaped motor generates high force and packages DNA fast. The mechanisms of subunit firing and motor coordination were poorly understood. As one can imagine, doping inactive subunit(s) in the motor may lead to defective drive force, velocity, and processivity. By observing the inactive subunit doped motors, we may deduct the coordination mechanism. Optical traps, a technique to study biological macromolecule dynamics at the single-molecule level, perfectly match the need to study viral genome packaging. I measured the packaging activity of wild-type T4 motors and inactive-subunit-doped T4 motors in the optical traps and analyzed the dynamic difference. I found that the packaging motor can tolerate inactive subunit(s). However, motors containing inactive subunit(s) exhibit slower packaging start, reduced packaging velocity, and increased pausing. These findings suggest the motor, by re-adjusting its grip on DNA, can skip an inactive subunit and resume DNA translocation, contrary to the strict coordination. Furthermore, I explored the fluorescently labeled T4 packaging assays in the optical traps. This assay allowed me to determine the number of inactive subunits in individual motors. However, due to multiple challenges, the measurement throughput has been too low to get sufficient data for further analysis. Optical trap users have suffered from unexpected fluorescence loss in the optical traps. Previous studies have shown that dye photobleaching is enhanced by absorption of visible fluorescence excitation plus infrared trap photons, a process that can be significantly reduced by minimizing simultaneous exposure to both light sources. Despite that, fluorescence emission of certain fluorophores near the trap focus can drop by 90% within 1 min. Hence, I explored another photobleaching pathway that results from direct excitation by the trapping laser alone. My results show that this trap-induced fluorescence loss is a two-photon absorption process, as demonstrated by a quadratic dependence on the intensity of the trapping laser. I further investigate how photostability is affected by the choice of dye molecule, excitation and emission wavelength, and labeled molecule. Finally, I discuss the different photobleaching pathways in combined trap-fluorescence measurements, which guide the selection of optimal dyes and conditions for more robust experimental protocols.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Physics

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2024

Author dc:creator

• Lu, Suoang

Contributors dc:contributor

• Chemla, Yann R
• Selvin, Paul R
• Aksimentiev, Aleksei
• Riedl, Caroline K

• Optical Trap
• Optical Tweezer
• Fluorescence
• Viral Packaging
• Bacteriophage T4
• Sinlge-molecule
• Photobleaching.

Statement dc:rights

• 2024 by Suoang Lu. All rights reserved.

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/127166

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F127166