{"doi":"10.1101/2025.09.02.673855","title":"Bacteriophage T4 gene 32 protein: Insights into its Interaction with ssDNA, binding cooperativity, and conformational change","abstract":"Abstract The single-stranded DNA binding protein of bacteriophage T4, gp32, has important roles in replication, recombination, and repair. gp32 possesses three domains: the central (core) domain which contains the binding trough for single-stranded DNA, the N-terminal domain, which interacts with the core domain of an adjacent ssDNA-bound protein, bringing about binding cooperativity, and the C-terminal domain, which interacts with other proteins involved in replication, recombination, and repair. The essential residues within the N-domain for the association with the adjacent DNA bound gp32, Lys-Arg-Lys-Ser-Thr, the “LAST Motif” , is almost identical to the ssDNA-interactive residues within the core domain binding trough, and was the basis of a model in which a “closed” ⇄ “open” conformational change within core domain controls DNA binding. In this study, we show that alteration of the core domain LAST sequence , while maintaining its composition , can have an effect on the binding parameters, and may be the result of a shift in the closed-open equilibrium. Additionally, utilizing a gp32 truncated at residue 227, as well as amino acid substituted variants, we have further localized the residues within the core domain responsible for the protein-protein association leading to cooperative ssDNA binding. Truncation leads to an increase in the non-cooperative affinity for single-stranded nucleic acids, which can be explained by the absence of a closed conformation in this variant. The truncated protein forms a tight complex with core domain on a 12-residue oligonucleotide, a potential candidate for further structural study.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":574222,"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":0.9594,"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":1482012,"name":"Michael P. Chapman","orcid":null,"position":1,"is_corresponding":false},{"id":1482013,"name":"Paul N. Brothers","orcid":null,"position":2,"is_corresponding":false},{"id":1482014,"name":"Shital Desai","orcid":null,"position":3,"is_corresponding":false},{"id":1071466,"name":"Richard L. Karpel","orcid":"0000-0003-1891-644X","position":4,"is_corresponding":false},{"id":1482011,"name":"Jules Guei","orcid":null,"position":0,"is_corresponding":true}],"reference_count":34,"raw_metadata":null,"created_at":"2026-07-19T02:57:40.686992Z","pmid":"40950117","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":[]}