{"doi":"10.1101/2020.10.29.361279","title":"Altering CLC stoichiometry by reducing non-polar side-chains at the dimerization interface","abstract":"ABSTRACT CLC-ec1 is a Cl - /H + antiporter that forms stable homodimers in lipid bilayers, with a free energy of −10.9 kcal/mole relative to the 1 subunit/lipid standard state in 2:1 POPE/POPG lipid bilayers. The dimerization interface is formed by four transmembrane helices: H, I, P and Q, that are lined by non-polar side-chains that come in close contact, yet it is unclear as to whether their interactions drive dimerization. To investigate whether non-polar side-chains are required for dimer assembly, we designed a series of constructs where side-chain packing in the dimer state is significantly reduced by making 4-5 alanine substitutions along each helix (H-ala, I-ala, P-ala, Q-ala). All constructs are functional and three purify as stable dimers in detergent micelles despite the removal of significant side-chain interactions. On the other hand, H-ala shows the unique behavior of purifying as a mixture of monomers and dimers, followed by a rapid and complete conversion to monomers. In lipid bilayers, all four constructs are monomeric as examined by single-molecule photobleaching analysis. Further study of the H-helix shows that the single mutation L194A is sufficient to yield monomeric CLC-ec1 in detergent micelles and lipid bilayers. X-ray crystal structures of L194A reveal the protein re-assembles to form dimers, with a structure that is identical to wild-type. Altogether, these results demonstrate that non-polar membrane embedded side-chains play an important role in defining dimer stability, but the stoichiometry is highly contextual to the solvent environment. Furthermore, we discovered that L194 is a molecular hot-spot for defining dimerization of CLC-ec1.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2020,"id":131501,"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":0.952,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":576231,"name":"Tuǧba N. Öztürk","orcid":"0000-0001-7225-9546","position":1,"is_corresponding":false},{"id":585531,"name":"Kunwoong Park","orcid":"0000-0002-5666-8486","position":2,"is_corresponding":false},{"id":585532,"name":"Hyun–Ho Lim","orcid":"0000-0002-5477-5640","position":3,"is_corresponding":false},{"id":356041,"name":"Janice Robertson","orcid":"0000-0002-5499-9943","position":4,"is_corresponding":false},{"id":585530,"name":"Kacey Mersch","orcid":"0000-0002-2497-1800","position":0,"is_corresponding":true}],"reference_count":50,"raw_metadata":null,"created_at":"2026-07-18T23:16:03.875886Z","pmid":null,"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":[]}