{"doi":"10.1002/pro.5220","title":"A pressure‐jump <scp>EPR</scp> system to monitor millisecond conformational exchange rates of spin‐labeled proteins","abstract":"Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) using nitroxide spin labels is a well-established technology for mapping site-specific secondary and tertiary structure and for monitoring conformational changes in proteins of any degree of complexity, including membrane proteins, with high sensitivity. SDSL-EPR also provides information on protein dynamics in the timescale of ps-μs using continuous wave lineshape analysis and spin lattice relaxation time methods. However, the functionally important time domain of μs-ms, corresponding to large-scale protein motions, is inaccessible to those methods. To extend SDSL-EPR to the longer time domain, the perturbation method of pressure-jump relaxation is implemented. Here, we describe a complete high-pressure EPR system at Q-band for both static pressure and ms-timescale pressure-jump measurements on spin-labeled proteins. The instrument enables pressure jumps both up and down from any holding pressure, ranging from atmospheric pressure to the maximum pressure capacity of the system components (~3500 bar). To demonstrate the utility of the system, we characterize a local folding-unfolding equilibrium of T4 lysozyme. The results illustrate the ability of the system to measure thermodynamic and kinetic parameters of protein conformational exchange on the ms timescale.","journal":"Protein Science","year":2024,"id":458824,"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":5,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9557,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":786645,"name":"Jason W. Sidabras","orcid":"0000-0002-4537-7501","position":1,"is_corresponding":false},{"id":498924,"name":"Christian Altenbach","orcid":"0009-0005-6902-6852","position":2,"is_corresponding":false},{"id":1285650,"name":"Jim R. Anderson","orcid":null,"position":3,"is_corresponding":false},{"id":1285651,"name":"Richard R. Mett","orcid":null,"position":4,"is_corresponding":false},{"id":787287,"name":"Robert A. Strangeway","orcid":null,"position":5,"is_corresponding":false},{"id":28549,"name":"James S. Hyde","orcid":"0000-0002-3023-1243","position":6,"is_corresponding":false},{"id":317676,"name":"Wayne L. Hubbell","orcid":"0000-0003-0577-3312","position":7,"is_corresponding":false},{"id":255552,"name":"Michael T. Lerch","orcid":"0000-0002-8173-4879","position":8,"is_corresponding":false},{"id":1285114,"name":"Julian D. Grosskopf","orcid":"0000-0003-4275-3628","position":0,"is_corresponding":true}],"reference_count":91,"raw_metadata":null,"created_at":"2026-07-19T02:03:50.608887Z","pmid":"39565088","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":[]}