{"doi":"10.1016/j.ab.2025.115859","title":"Integrative biophysical characterization of molecular interactions: A case study with the sfGFP–nanobody complex","abstract":"This study compares several analytical biophysical methods for investigating protein-protein interactions (PPIs) in solution, using the interaction between superfolder green fluorescent protein (sfGFP) and its anti-sfGFP nanobody enhancer as a model system. Techniques evaluated include microscale thermophoresis, fluorescence correlation spectroscopy, analytical ultracentrifugation with multi-wavelength and fluorescence detection, isothermal titration calorimetry, and analytical size exclusion chromatography coupled to multi-angle static light scattering and dynamic light scattering. Each method was assessed for information content, dynamic range, precision, and complementarity. The results consistently indicate a single-digit nanomolar dissociation constant and 1:1 stoichiometry for the interaction. While each technique offers unique insights into binding affinity, thermodynamics, and stoichiometry of the interaction, the multi-method approach provides a more complete and reliable characterization of PPIs. The study demonstrates how combining multiple complementary techniques enhances the robustness of PPI analysis in solution-phase conditions.","journal":"Analytical Biochemistry","year":2025,"id":543666,"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":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9542,"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":1433657,"name":"James Bosco","orcid":"0009-0006-7141-8705","position":1,"is_corresponding":false},{"id":378100,"name":"Matthew J. Sydor","orcid":"0000-0002-8738-4451","position":2,"is_corresponding":false},{"id":1069710,"name":"Michelle D. Nemetchek","orcid":"0000-0003-3524-952X","position":3,"is_corresponding":false},{"id":1128724,"name":"Saeed Mortezazadeh","orcid":"0000-0001-6437-6953","position":4,"is_corresponding":false},{"id":1433658,"name":"Liam Kerr","orcid":"0000-0002-5459-4639","position":5,"is_corresponding":false},{"id":1404394,"name":"Scott M. Bird","orcid":"0009-0007-1629-5762","position":6,"is_corresponding":false},{"id":1433997,"name":"Roza Gabdullina","orcid":null,"position":7,"is_corresponding":false},{"id":499202,"name":"Cindee Yates-Hansen","orcid":"0000-0001-8116-2586","position":8,"is_corresponding":false},{"id":499200,"name":"Levi J. McClelland","orcid":"0000-0002-0868-8925","position":9,"is_corresponding":false},{"id":366905,"name":"Ekaterina Voronina","orcid":"0000-0002-0194-4260","position":10,"is_corresponding":false},{"id":301766,"name":"Trushar R. Patel","orcid":"0000-0003-0627-2923","position":11,"is_corresponding":false},{"id":399433,"name":"Borries Demeler","orcid":"0000-0002-2414-9518","position":12,"is_corresponding":false},{"id":598977,"name":"Aysha K. Demeler","orcid":null,"position":0,"is_corresponding":true}],"reference_count":47,"raw_metadata":null,"created_at":"2026-07-19T02:53:08.338070Z","pmid":"40204056","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":[]}