{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T11:02:55Z","timestamp":1791198175863,"version":"4.1.0"},"reference-count":43,"publisher":"AIP Publishing","issue":"13","license":[{"start":{"date-parts":[[2026,10,3]],"date-time":"2026-10-03T00:00:00Z","timestamp":1790985600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100006224","name":"Argonne National Laboratory","doi-asserted-by":"publisher","award":["DE-AC02-06CH11357"],"award-info":[{"award-number":["DE-AC02-06CH11357"]}],"id":[{"id":"10.13039\/100006224","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["P41 GM118217"],"award-info":[{"award-number":["P41 GM118217"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000062","name":"National Institute of Diabetes and Digestive and Kidney Diseases","doi-asserted-by":"publisher","award":["Intramural Research Program"],"award-info":[{"award-number":["Intramural Research Program"]}],"id":[{"id":"10.13039\/100000062","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["pubs.aip.org"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2026,10,7]]},"abstract":"<jats:p>Understanding how biomolecules execute their functions requires knowledge not only of their static ground-state structures but also of the conformational dynamics, solvation, and intermolecular interactions that they sample under physiological conditions. High-resolution crystallography, the dominant source of atomic-resolution structures, captures the molecule within a rigid lattice that can favor particular conformations and is largely silent on the structural dynamics present in solution. Here, we develop temperature-dependent small- and wide-angle x-ray scattering as a probe of biomolecular structure, dynamics, and interactions in the solution phase and apply it to two well-characterized model proteins: the Villin headpiece subdomain and the GB3 domain. Concentration-dependent measurements over temperatures spanning supercooled (\u221216\u2009\u00b0C) to fully unfolded (120\u2009\u00b0C), extrapolated to infinite dilution, allow us to recover the forward-scattering intensity I0(T), the radius of gyration Rg(T), and the real-space pair-distribution function p(r,T). Both Villin and GB3 exhibit at least two structurally distinct folded conformations and, when unfolded at high concentration (\u223c20\u00a0mg\/ml), form transient dimers due to entanglement of the disordered peptide chains. Villin exhibits concentration-dependent dimerization of its folded structure and an anomalous loss of scattering power upon unfolding that we hypothesize arises from release of bound chlorides. Subtracting the fully disordered, high-temperature ensemble represented in p(r,Tmax) from all other curves generates \u0394p(r,T) with sufficient spatial resolution to track the temperature dependence of secondary and tertiary structural features. These real-space fingerprints of the folds place stringent constraints on candidate atomic structures and may provide a solution-phase route to atomic-level understanding of biomolecular structure and dynamics under the physiological conditions in which they function.<\/jats:p>","DOI":"10.1063\/5.0350696","type":"journal-article","created":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T10:39:46Z","timestamp":1791196786000},"update-policy":"https:\/\/doi.org\/10.1063\/aip-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Probing protein structures in solution via concentration- and temperature-dependent small- and wide-angle x-ray scattering"],"prefix":"10.1063","volume":"165","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8871-3886","authenticated-orcid":false,"given":"Hyun Sun","family":"Cho","sequence":"first","affiliation":[{"name":"Laboratory of Chemical Physics, NIDDK, National Institutes of Health , Bethesda, Maryland 20892,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7225-9212","authenticated-orcid":false,"given":"Friedrich","family":"Schotte","sequence":"additional","affiliation":[{"name":"Laboratory of Chemical Physics, NIDDK, National Institutes of Health , Bethesda, Maryland 20892,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0052-1899","authenticated-orcid":false,"given":"John M.","family":"Louis","sequence":"additional","affiliation":[{"name":"Laboratory of Chemical Physics, NIDDK, National Institutes of Health , Bethesda, Maryland 20892,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8261-0624","authenticated-orcid":false,"given":"Philip","family":"Anfinrud","sequence":"additional","affiliation":[{"name":"Laboratory of Chemical Physics, NIDDK, National Institutes of Health , Bethesda, Maryland 20892,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"317","published-online":{"date-parts":[[2026,10,5]]},"reference":[{"issue":"5038","key":"2026100506394460200_c1","doi-asserted-by":"publisher","first-page":"1598","DOI":"10.1126\/science.1749933","article-title":"The energy landscapes and motions of proteins","volume":"254","year":"1991","journal-title":"Science"},{"issue":"7172","key":"2026100506394460200_c2","doi-asserted-by":"publisher","first-page":"964","DOI":"10.1038\/nature06522","article-title":"Dynamic personalities of proteins","volume":"450","year":"2007","journal-title":"Nature"},{"issue":"11","key":"2026100506394460200_c3","doi-asserted-by":"publisher","first-page":"789","DOI":"10.1038\/nchembio.232","article-title":"The role of dynamic conformational ensembles in biomolecular recognition","volume":"5","year":"2009","journal-title":"Nat. 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