QUANTIFYING OLIGOMERIC STATES OF PROTEINS IN-CELL VIA SINGLE-MOLECULE SUPER-RESOLUTION MICROSCOPY

Modifying protein oligomeric stoichiometry plays a vital role in initiating signaling pathways, transcriptional regulation, and cell apoptosis regulation. However, the quantification of their oligomeric states in cells is technically challenging. This dissertation describes our efforts in developing in-cell single-molecule imaging assays to quantify oligomeric states of membrane and cytosolic proteins. Quantifying oligomeric states of highly-expressed membrane proteins were achieved using protein localizations obtained from super-resolution microscopy. A single-molecule assay was developed to extract the surface protein oligomer populations using the probability density function of molecule density (PDFMD). We provided the theoretical model of PDFMD, further validated the method using simulated single-molecule fluorescent movies, and applied it to two membrane proteins, UhpT and SbmA, in E. coli cells. While PDFMD is suitable for membrane proteins, direct application of PDFMD to cytosolic proteins is not feasible due to their heterogeneous intracellular distributions. To quantify oligomeric stoichiometry of cytosolic proteins, we established a single-molecule Förster resonance energy transfer (smFRET) based method. We investigated the dimerization of SOD1 by imaging SOD1-Halo-tag proteins conjugated with FRET donor fluorophore, PA-JF549, and acceptor fluorophore, JF646, in COS7 cells. Analyzing the FRET contribution on donor's intensity distributions allows us to estimate the dimerization of SOD1 proteins in cells. To apply our methods to a system that can provide physiologically relevant information, we further used CRISPR-Cas9 to generate H1 hESC lines, where the SOD1 gene with genetically tagged with SNAP-tag, allowing us to dissect the oligomeric states of SOD1 at an endogenous expression level. Further, the ability to differentiate hESC to different cell types (e.g., hepatocytes or neurons) allows us to explore the roles of oligomeric states on cellular processes in a cell-specific manner.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Houston

Year dc:date.issued

2022

Author dc:creator

• Xie, Xihong

Advisor dc:contributor.advisor

• Chen, Tai-Yen

Committee members dc:contributor.committeemember

• Baldelli, Steven
• Xu, Shoujun
• Cai, Chengzhi
• Eriksen, Jason

• Protein oligomeric stoichiometry
• Dimerization
• Single-molecule imaging

Statement dc:rights

• The author of this work is the copyright owner. UH Libraries and the Texas Digital Library have their permission to store and provide access to this work. UH Libraries has secured permission to reproduce any and all previously published materials contained in the work. Further transmission, reproduction, or presentation of this work is prohibited except with permission of the author(s).

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/10657/13330

OAI identifier oai:identifier

oai:uh-ir.tdl.org:10657/13330