Single-Molecule Quantification Methods To Detect Protein Oligomeric Stoichiometry In Cell

Protein oligomerization plays a critical role in many biological processes, but studying its quantification in cells has been challenging. In this dissertation, we developed three intracellular single-molecule methodologies based on super-resolution fluorescence microscopy to probe the oligomeric stoichiometry of cytosolic and membrane proteins. Using these techniques, we investigated the oligomeric modification of Cu/Zn superoxide dismutase (SOD1) under different conditions. We found that treating cells with hydrogen peroxide (H2O2) promotes SOD1 dimerization and decreases cellular viscosity. Additionally, we observed that SOD1 tends to monomerize under reduced and Cu-depleted conditions. Furthermore, we developed a location-based assay, probability of neighbor density (PND), which can quantify protein oligomeric stoichiometry up to trimer. Our studies provide new insights into the molecular mechanisms underlying protein oligomerization and highlight the potential of these methodologies for future research in this field. In the first chapter, we developed a single-molecule fluorescence anisotropy (smFA) assay to study the oligomeric modification of SOD1 in response to H2O2. The smFA assay was validated using monomeric and dimeric mEos4b, and then it was performed in live COS7 cells overexpressing SOD1-mEos4b. We found that treating cells with H2O2 promoted SOD1 dimerization and reduced cellular viscosity in 2 h. However, after 24 h, the cells returned to a steady state similar to the basal state. We further developed a single-molecule Förster resonance energy transfer (smFRET) assay to quantitatively investigate the oligomeric state of SOD1 under different conditions. Firstly, we observed that 70% of SOD1 existed as dimer under basal condition. Interestingly, SOD1 tended to monomerize under both reduced and Cu-depleted conditions. However, under Cu-stressed conditions, no SOD1 monomer was detected. Additionally, under oxidative conditions, the SOD1 monomer component decreased to approximately 20%. In the last chapter, we introduced the PND assay, which links spatial information with protein oligomeric stoichiometry, up to the trimer level. Firstly, we developed a theoretical description of PND for pure oligomers, which was validated using simulated ground truth data. Since proteins can exist in mixed oligomeric states, we successfully extended the theoretical model to account for mixed stoichiometry. Furthermore, we demonstrated the assay's ability to accurately quantify protein levels with negligible error.