{"doi":"10.1093/nar/gkaf1366","title":"Revealing hidden protonated conformational states in RNA dynamic ensembles","abstract":"Identifying protonated states within RNA ensembles, quantifying their pKas, and elucidating the kinetic mechanisms by which they form is essential for understanding protonation-coupled biochemical reactions and how RNAs sense and adapt to pH fluctuations. However, detecting protonated states is challenging when they are short-lived and lowly populated. Here, using pH-dependent NMR chemical exchange, kinetic solvent isotope effects, and mutation, we show that a low-populated (0.4% at pH 6.4) conformational state of HIV-1 TAR RNA is coupled to protonation of a C⁺-C mismatch. Despite an intrinsic pKa of ~7.1, the energetic penalty to form this alternative conformation depressed the apparent pKa to ∼4.0, below the pH range typically probed experimentally. Substituting C-C with a G-C base pair abolished the pH-dependence of these dynamics, confirming C-C as the protonation site. This hidden protonated state competes with a more abundant conformation harboring a C-A⁺ mismatch, producing a non-monotonic ensemble response to pH. Both transitions follow an induced-fit mechanism, in which solvent-exposed nucleobases are rapidly protonated followed by slower changes in secondary structure. These findings reveal a general mechanism for protonation-coupled conformational switching in RNA and provide a framework for dissecting sparsely populated protonated states and their multi-protonation-state dynamics.","journal":"Nucleic Acids Research","year":2025,"id":549598,"datarank":0.16479184330021646,"base_score":1.0986122886681096,"endowment":1.0986122886681096,"self_citation_contribution":0.16479184330021646,"citation_network_contribution":0.0,"self_endowment_contribution":0.16479184330021646,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.958,"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":991255,"name":"Rohit Roy","orcid":"0000-0001-8569-3245","position":1,"is_corresponding":false},{"id":467477,"name":"Laura R. Ganser","orcid":"0000-0002-7357-4588","position":2,"is_corresponding":false},{"id":1391489,"name":"Linshu Li","orcid":null,"position":3,"is_corresponding":false},{"id":259604,"name":"Hashim M. Al‐Hashimi","orcid":"0000-0003-0681-1747","position":4,"is_corresponding":false},{"id":991256,"name":"Ainan Geng","orcid":"0000-0002-0271-4141","position":0,"is_corresponding":true}],"reference_count":92,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:54:07.823422Z","pmid":"41414670","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":[]}