Probing Oxidant Effects on Superoxide Dismutase 1 Oligomeric States in Live Cells Using Single-Molecule Fluorescence Anisotropy

Abstract

The protein Cu/Zn superoxide dismutase (SOD1) is known to function as a dimer, but its concentration in cells (∼50 μM) and the dimerization constant (K d of 500 μM) results suggest that it exists in a monomer-dimer equilibrium. It is unclear how the oligomeric state of SOD1 changes when cells are initially exposed to high levels of extracellular oxidative stress. To address this problem, we introduced the single-molecule fluorescence anisotropy (smFA) assay to explore SOD1 oligomeric states in live COS7 cells. smFA specifically probes the fluorescence polarization changes caused by molecular rotations where the fast-rotating molecules (either due to smaller hydrodynamic volume or less viscous environments) deteriorate the emission polarization and thus lower the anisotropy. After validating that smFA is effective in distinguishing monomeric and dimeric fluorescence proteins, we overexpressed SOD1 in live COS7 cells and investigated how its oligomeric state changes under basal, 2 h, and 24 h 100 μM H 2 O 2 treatments. We found that treating cells with H 2 O 2 promotes SOD1 dimerization and decreases cellular viscosity in 2 h. Interestingly, prolonged H 2 O 2 treatments show similar results as the basal conditions, indicating that cells return to a steady state similar to the basal state after 24 h, despite the presence of H 2 O 2 . Our results demonstrate that SOD1 changes its oligomeric state equilibrium in response to extracellular oxidative stresses. smFA will open new opportunities to explore the relationship between the SOD1 oligomer state and its H 2 O 2 -based signaling and transcription regulation roles.

Advancements and Practical Considerations for Biophysical Research: Navigating the Challenges and Future of Super-resolution Microscopy

“…Highlighting the power of single-molecule approaches in dynamic studies, our work contributes two new methodologies: single-molecule fluorescence anisotropy (smFA) and the single-molecule neighboring density (smND) assay. , smFA brings a new dimension to SRM by enabling the observation of intracellular viscosity through the measurement of rotational mobility of fluorescent proteins. On the other hand, the smND assay quantifies protein oligomeric states directly within cells by analyzing the spatial distribution and density of individual protein molecules . Utilizing the probability of neighbor density model, which accounts for factors like protein concentrations and photoactivation efficiency, the smND assay can distinguish between various oligomeric states by comparing experimental data against simulated distributions of protein assemblies.…”

Dual-Functional Fluorescent Probe in the Diagnosis of Liver Injury and the Evaluation of Drug Therapy with Double Signal Amplification

“…In certain instances, the condition only becomes discernible when patients manifest evident complications, frequently resulting in the missed opportunity for an optimal diagnosis. Additionally, conventional diagnostic methods still have limitations, such as low specificity, , cumbersome processes, and notable risks. , Recently, fluorescence optical imaging and analysis techniques have played a pivotal role in early disease diagnosis, treatment, new drug development, and drug delivery, , owing to noninvasive nature, high sensitivity, and simple operation. − Hence, the development of an appropriate fluorescence probe to elucidate the mechanisms, classification, and the biological and pathological functions related to diabetes and associated diseases with viscosity and polarity holds profound significance. , …”

From Monomers to Hexamers: A Theoretical Probability of the Neighbor Density Approach to Dissect Protein Oligomerization in Cells

“…Ensemble approaches, such as ensemble FRET, BiFC, FCS, and PLA, served as foundational pillars, providing insights into the predominant oligomeric states within cellular populations. − However, while these methods elucidated average oligomeric states, they could often overlook molecular heterogeneity or rare oligomeric forms. To unmask this concealed diversity, single-molecule techniques like smFRET, single particle tracking, and single-molecule anisotropy were adopted. ,− These methods illuminated the full spectrum of oligomeric states by capturing individual protein molecules, revealing not just the dominant forms but also transient or less abundant oligomeric states.…”

“…The complex interplay of proteins coming together to form larger structuresknown as oligomerizationhas far-reaching effects on how cells function and maintain their stability. − Research has consistently shown that proteins often act in their combined or “oligomeric” forms for key cellular processes, such as regulating genes and controlling enzyme activity. − The behavior of these protein assemblies is not solely determined by their individual properties but is also shaped by the specific conditions within the cell. − While in vitro studies provide immensely valuable information, they often fail to fully capture the true essence of protein behaviors in a cellular context. For example, they might miss the effects of changes in cellular components or neglect variables like protein concentration and post-translational modifications.…”