{"doi":"10.1016/j.bidere.2025.100061","title":"Heptad insertion extends the E. coli ROP protein by 50 %","abstract":"Antibody function involves conformational variability, yet their extreme flexibility complicates measurement of their structure and properties. They also have numerous ligands, suggesting that a rigid bivalent ligand construct of appropriate length could interact with an antibody to reduce its flexibility for imaging, measurement or functional effect. Such a construct would ideally have an inter-ligand spacer that is fairly rigid and with length between 6 and 10 nm. Coiled-coil bundles of alpha helices represent a common motif in protein structure whose relatively simple parallel geometry makes them suitable for rational modification including applications in metrology. In this study, we describe a heptad-insertion heuristic for extending bundles and apply it to the E. coli ROP/ROM protein, which is a 13 kDa, thermostable RNA-binding unit that is naturally a 4-helix dimer and has been engineered to self-associate in various ways to form larger assemblies. We first introduced a tryptophan residue into the core (wild-type lacks tryptophan) to support precise quantitation, and then extended the protein to 150 % of its native length by inserting four helical heptads. We report the engineering process and crystal structures of the tryptophan mutant and the lengthened protein, which also contains a new phenylalanine in the core. Where wild-type has a length of 4.3 nm, the extended variant has length 6.5 nm and may serve as a rigid module for higher order constructs.","journal":"BioDesign Research","year":2025,"id":584587,"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":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.962,"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":299793,"name":"D. Travis Gallagher","orcid":"0000-0003-1386-7255","position":1,"is_corresponding":false},{"id":1497747,"name":"Ioannis Karageorgos","orcid":"0000-0002-2799-766X","position":0,"is_corresponding":true}],"reference_count":33,"raw_metadata":null,"created_at":"2026-07-19T02:59:16.166424Z","pmid":"42038005","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":[]}