{"doi":"10.1016/j.jlr.2025.100846","title":"Nanodisc single-molecule pulldown to study lipid-protein interactions","abstract":"Beyond serving structural roles in the cell membrane, many phospholipids, including phosphatidylinositol phosphates (PIPs), are key signaling molecules that regulate a myriad of cellular processes. Specific interactions with PIPs are crucial for the functions of many signaling proteins, highlighting the need for a convenient and robust method to study lipid-protein interactions. Previously we established a fluorescence microscopy-based lipid single-molecule pulldown (lipid-SiMPull) assay for detecting interactions between fluorescently tagged proteins of interest in whole-cell lysates and small unilamellar vesicles (SUVs) containing phospholipids of interest. Despite unique advantages of the lipid-SiMPull assay, SUV is not an optimal membrane model due to its instability, heterogeneity in size, and a membrane curvature inconsistent with the relative flatness of the cell membrane. Here we report the use of lipid Nanodiscs in lipid-SiMPull. Using PIP-protein pairs of known interactions, we show that Nanodiscs containing various PIPs can pull down protein targets specifically, with an estimated detection threshold of K d in the 10-20 μM range. Remarkably, we find that each Nanodisc is bound by one copy of the protein (or protein dimer), conferring true single-molecule resolution to the assay. Transient interactions are characterized by the re-binding of proteins to individual Nanodiscs, and dissociation rates ( k off ) are determined from dwell time analysis. We apply this assay to interrogate structural requirements for the stability of AKT binding of PI(3,4,5)P 3 . Our results suggest that an intra-molecular interaction between the PH domain and kinase domain is critical for stabilizing the AKT-PI(3,4,5)P 3 interaction.","journal":"Journal of Lipid Research","year":2025,"id":528371,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9609,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1132093,"name":"Shweta Shree","orcid":null,"position":1,"is_corresponding":false},{"id":436590,"name":"Nilmani Singh","orcid":"0000-0002-3684-7022","position":2,"is_corresponding":false},{"id":383949,"name":"Stephen G. Sligar","orcid":"0000-0002-5548-2866","position":3,"is_corresponding":false},{"id":436593,"name":"Jie Chen","orcid":"0000-0002-7887-3747","position":4,"is_corresponding":false},{"id":569809,"name":"Adriana Reyes‐Ordoñez","orcid":"0000-0003-3482-3841","position":0,"is_corresponding":true}],"reference_count":34,"raw_metadata":null,"created_at":"2026-07-19T02:50:48.492873Z","pmid":"40545238","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}