{"doi":"10.1101/2025.08.29.672704","title":"Optimal TELSAM-Target Protein Linker Character is Target Protein-Dependent","abstract":"Abstract Fusing a variant of the sterile alpha motif domain of the human translocation ETS leukaemia protein (TELSAM) to a protein of interest has been shown to significantly enhance crystallization propensity. TELSAM is a pH-dependent, polymer-forming protein crystallization chaperone which, when covalently fused to a protein of interest, forms a stable, well-ordered crystal lattice. However, despite its success, a challenge persists in that crystal quality and diffraction limits appear to be heavily dependent on the choice of linker between TELSAM and the protein of interest, with identification of a functional linker relying on trial-and-error methods. Likewise, previous studies revealed that the 10xHis tag at the TELSAM N-terminus can either facilitate or hinder the ordered crystallization of target proteins attached via flexible or semi-flexible linkers. To address these challenges, we designed multiple constructs with several types of linkers—rigid (helical fusion), semi-flexible (Pro-Ala n ), and flexible (poly-Gly)—of varying lengths to fuse a designed ankyrin repeat protein (DARPin) to the TELSAM C-terminus. Semi-flexible and flexible linker constructs were made with and without the 10xHis tag. Our findings indicate that short semi-flexible and rigid linkers consistently yield large crystals within 24 hours with a DARPin target protein, but that flexible linkers perform best with a TNK1 UBA domain target protein. Removing the 10xHis tag enhanced crystallization rates, improved crystal morphology, and increased the crystallization propensity of semi-flexible and flexible linker constructs. While removing the His tag did not have a significant effect on crystal size, it improved the diffraction limits and crystal quality of the 1TEL-PA-DARPin construct. These results suggest that the ideal linker selection primarily depends on the properties of the target protein. Our data support the recommendation to use a short yet flexible or semi-flexible linker between TELSAM and the target protein to facilitate protein crystallization and high-resolution structure determination. Synopsis In this study, we examine the effect of short to medium-length flexible, semi-flexible, and rigid linkers on the crystallization of a DARPin fused to the 1TEL protein crystallization chaperone, demonstrating that while rigid linkers impair crystallization and reduce diffraction quality, the ideal linker character remain target-protein dependent.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":573939,"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":0.9569,"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":1481457,"name":"Alihikaua Keliiliki","orcid":null,"position":1,"is_corresponding":false},{"id":1146691,"name":"Jacob Averett","orcid":"0000-0002-2287-1590","position":2,"is_corresponding":false},{"id":1481057,"name":"Joseph Gonzalez","orcid":"0000-0002-7527-7616","position":3,"is_corresponding":false},{"id":1481458,"name":"Ethan Noakes","orcid":null,"position":4,"is_corresponding":false},{"id":1146692,"name":"E.F. 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Hansen","orcid":null,"position":8,"is_corresponding":false},{"id":1481059,"name":"Riley Nickles","orcid":"0009-0009-5570-9096","position":9,"is_corresponding":false},{"id":1481060,"name":"Miles Bradford","orcid":"0009-0004-8380-4975","position":10,"is_corresponding":false},{"id":1481461,"name":"Сара Солеймани","orcid":null,"position":11,"is_corresponding":false},{"id":828535,"name":"Tobin Smith","orcid":"0000-0003-1766-4738","position":12,"is_corresponding":false},{"id":828527,"name":"Supeshala Nawarathnage","orcid":"0000-0002-8850-6625","position":13,"is_corresponding":false},{"id":1481462,"name":"Prasadika Samarwickrama","orcid":null,"position":14,"is_corresponding":false},{"id":1481463,"name":"Ariel Kelsch","orcid":null,"position":15,"is_corresponding":false},{"id":828533,"name":"Derick Bunn","orcid":"0000-0002-0005-5023","position":16,"is_corresponding":false},{"id":828534,"name":"Cameron Stewart","orcid":"0000-0002-2786-6256","position":17,"is_corresponding":false},{"id":1124607,"name":"Wisdom Abiodun","orcid":"0000-0003-0312-2858","position":18,"is_corresponding":false},{"id":1124609,"name":"Evan Tsubaki","orcid":"0009-0001-8014-9643","position":19,"is_corresponding":false},{"id":828537,"name":"Seth Brown","orcid":"0000-0003-2888-7208","position":20,"is_corresponding":false},{"id":364771,"name":"Tzanko Doukov","orcid":"0000-0001-8625-2572","position":21,"is_corresponding":false},{"id":694978,"name":"James Moody","orcid":"0000-0003-2266-5348","position":22,"is_corresponding":false},{"id":828536,"name":"Maria J. Pedroza Romo","orcid":"0000-0002-7441-3874","position":0,"is_corresponding":true}],"reference_count":20,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:57:36.753600Z","pmid":"40950120","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":[]}