SiMPull-POP: Quantification of Membrane Protein Assembly via Single Molecule Photobleaching.

Traditional methods for studying protein-protein interactions often lack the resolution to quantitatively distinguish distinct oligomeric states, particularly for membrane proteins within their native lipid environments. To address this limitation, we developed SiMPull-POP (single-molecule pull-down polymeric nanodisc photobleaching), a single-molecule technique designed to quantify membrane protein oligomerization with high sensitivity and in a near-native context. The goal of SiMPull-POP is to enable precise, quantitative analysis of membrane protein assembly by preserving native lipid interactions using diisobutylene maleic acid (DIBMA) to form nanodiscs. Unlike ensemble methods such as co-immunoprecipitation or FRET, which average out heterogeneous populations, SiMPull-POP uses photobleaching to resolve monomeric, dimeric, and higher-order oligomeric states at the single-molecule level. We validated SiMPull-POP using several model systems. A truncated, single-pass transmembrane protein (Omp25) appeared primarily monomeric, while a membrane-tethered FKBP protein exhibited ligand-dependent dimerization upon addition of the AP ligand. Applying SiMPull-POP to EphA2, a receptor tyrosine kinase, we found it to be mostly monomeric in the absence of its ligand, Ephrin-A1, and shifting toward higher-order oligomers upon ligand binding. To explore factors influencing ligand-independent assembly, we modulated membrane cholesterol content. Reducing cholesterol induced spontaneous EphA2 oligomerization, indicating that cholesterol suppresses receptor self-association. Overall, SiMPull-POP offers significant advantages over conventional techniques by enabling quantitative, single-molecule resolution of membrane protein complexes in a native-like environment. This approach provides critical insights into how membrane properties and external stimuli regulate protein assembly, supporting broader efforts to understand membrane protein function in both normal and disease states. Key features • Precise determination of membrane protein stoichiometry (e.g., monomer, dimer, oligomer) by directly counting photobleaching steps, overcoming the averaging limitations of bulk assays. • By incorporating membrane proteins into DIBMA lipid particles (DIBMALPs), this preserves native lipid interactions, offering a more physiologically relevant context for studying protein assembly. • Sensitively detects ligand-induced or membrane property-driven changes in oligomerization, making it a powerful tool for investigating both constitutive and regulated protein interactions.