{"doi":"10.1021/acs.jpclett.4c03247","title":"Determination of Thiol Protonation States by Sulfur X-ray Spectroscopy in Biological Systems","abstract":"Cysteine is one of the most functionally diverse of the proteinogenic amino acids, owing to its reactive thiol side chain that can undergo deprotonation to form a strongly nucleophilic thiolate. However, few techniques can directly interrogate sulfur charge and covalency in cysteine, particularly in proteins. X-ray spectroscopies provide an element specific probe of sulfur. We demonstrate the sensitivity of S Kβ and Kα X-ray emission spectroscopy (XES) to cysteine ionization and compare it to S K-edge X-ray absorption spectroscopy (XAS) in the physiologically relevant biomolecules l -cysteine and N -acetyl- l -cysteine at room temperature in solution phase. Kβ XES and K-edge XAS are most sensitive to chemical changes at the cysteine thiol and can be used to evaluate the composition of thiol/thiolate mixtures. These results provide a foundation for assessing the p K a of functionally significant cysteine residues in proteins and open the door to time-resolved studies of cysteine-dependent enzymes.","journal":"The Journal of Physical Chemistry Letters","year":2025,"id":531153,"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":4,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9572,"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":383627,"name":"Alec H. Follmer","orcid":"0000-0002-6244-6804","position":1,"is_corresponding":false},{"id":299425,"name":"Jeffrey T. Babicz","orcid":"0000-0002-1476-8390","position":2,"is_corresponding":false},{"id":409833,"name":"Victor Sosa Alfaro","orcid":"0000-0001-8149-5137","position":3,"is_corresponding":false},{"id":628101,"name":"Ryan G. Hadt","orcid":"0000-0001-6026-1358","position":4,"is_corresponding":false},{"id":282896,"name":"Mark S. Hunter","orcid":"0000-0002-0110-7075","position":5,"is_corresponding":false},{"id":388663,"name":"Mark A. Wilson","orcid":"0000-0001-6317-900X","position":6,"is_corresponding":false},{"id":414298,"name":"Dimosthenis Sokaras","orcid":"0000-0001-8117-1933","position":7,"is_corresponding":false},{"id":240879,"name":"Roberto Alonso‐Mori","orcid":"0000-0002-5357-0934","position":8,"is_corresponding":false},{"id":1242422,"name":"Ryan D. Ribson","orcid":"0000-0002-3755-5777","position":0,"is_corresponding":true}],"reference_count":43,"raw_metadata":null,"created_at":"2026-07-19T02:51:10.077559Z","pmid":"40012333","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":[]}