{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T13:25:32Z","timestamp":1787059532948,"version":"build-2736575974"},"reference-count":47,"publisher":"American Chemical Society (ACS)","issue":"16","license":[{"start":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T00:00:00Z","timestamp":1786320000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T00:00:00Z","timestamp":1786320000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T00:00:00Z","timestamp":1786320000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/501100001412","name":"Council of Scientific and Industrial Research, India","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001412","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100007488","name":"Indian Institute of Technology Delhi","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100007488","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004541","name":"Ministry of Education, India","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100004541","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,8,18]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Escherichia coli methionine adenosyltransferase (MetK) catalyzes the synthesis of S-adenosylmethionine from ATP and l-methionine and is essential for cellular metabolism. To gain insight into its conformational stability and folding behavior in vitro, we investigated the unfolding and refolding of MetK using guanidine hydrochloride (GdnHCl), urea, and acid (pH 2.0) as denaturing conditions. Unfolding was monitored using enzymatic activity, tryptophan fluorescence, far-UV circular dichroism (CD), native-PAGE, and light scattering. GdnHCl, urea, and acid treatment generated structurally distinct denatured ensembles with different structural and functional properties. Although unfolding transitions monitored individually by tryptophan fluorescence and far-UV CD could be described by simple unfolding models, noncoincident transitions together with singular value decomposition (SVD) analysis suggested conformational heterogeneity during unfolding. Refolding of denatured MetK in native buffer was inefficient and accompanied by aggregation, whereas the inclusion of physiologically relevant osmolytes such as glycerol and trehalose substantially improved refolding yields. Complete recovery of enzymatic activity was achieved only from the 8 M urea-denatured state, with a refolding half-time of 4.64 \u00b1 0.23 h. Collectively, these results reveal denaturant-dependent differences in the unfolding and refolding behavior of MetK and highlight the importance of unfolded-state properties and osmolyte-assisted stabilization in determining productive folding outcomes.<\/jats:p>","DOI":"10.1021\/acs.biochem.6c00531","type":"journal-article","created":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T07:01:54Z","timestamp":1786345314000},"page":"2522-2534","source":"Crossref","is-referenced-by-count":0,"title":["Spontaneous\nRefolding of\n                    <i>Escherichia coli<\/i>\n                    Methionine Adenosyltransferase (MetK) Is Extremely\nSlow, Indicative of Chaperone Dependence"],"prefix":"10.1021","volume":"65","author":[{"given":"Anupam","family":"Barai","sequence":"first","affiliation":[{"name":"Indian Institute of Technology Delhi , , , ,","place":["Hauz Khas, New Delhi, India, 110016"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pronoy","family":"Majumder","sequence":"additional","affiliation":[{"name":"Indian Institute of Technology Delhi , , , ,","place":["Hauz Khas, New Delhi, India, 110016"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Madhurima","family":"Sarkar","sequence":"additional","affiliation":[{"name":"Indian Institute of Technology Delhi , , , ,","place":["Hauz Khas, New Delhi, India, 110016"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3668-1640","authenticated-orcid":true,"given":"Tapan K.","family":"Chaudhuri","sequence":"additional","affiliation":[{"name":"Indian Institute of Technology Delhi , , , ,","place":["Hauz Khas, New Delhi, India, 110016"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,10]]},"reference":[{"issue":"4096","key":"2026081808124013900_cit1","doi-asserted-by":"publisher","first-page":"223","DOI":"10.1126\/science.181.4096.223","article-title":"Principles\nThat Govern the Folding of Protein Chains","volume":"181","author":"Anfinsen","year":"1973","journal-title":"Science"},{"issue":"6968","key":"2026081808124013900_cit2","doi-asserted-by":"publisher","first-page":"884","DOI":"10.1038\/nature02261","article-title":"Protein\nFolding and Misfolding","volume":"426","author":"Dobson","year":"2003","journal-title":"Nature"},{"issue":"2","key":"2026081808124013900_cit3","doi-asserted-by":"publisher","first-page":"253","DOI":"10.1016\/j.molcel.2010.10.006","article-title":"The Heat Shock\nResponse: Life on\nthe Verge of Death","volume":"40","author":"Richter","year":"2010","journal-title":"Mol. 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Bacteriol."},{"issue":"5","key":"2026081808124013900_cit7","doi-asserted-by":"publisher","first-page":"909","DOI":"10.1016\/0092-8674(93)90470-B","article-title":"Folding in Vivo of Bacterial Cytoplasmic\nProteins:\nRole of GroEL","volume":"74","author":"Horwich","year":"1993","journal-title":"Cell"},{"key":"2026081808124013900_cit8","doi-asserted-by":"publisher","first-page":"446","DOI":"10.1007\/BF00333275","article-title":"A DNA Fragment Containing\nthe groE Genes Can Suppress Mutations in the Escherichia coli dnaA Gene","volume":"202","author":"Jenkins","year":"1986","journal-title":"Mol. Gen. 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Microbiol."},{"issue":"5","key":"2026081808124013900_cit15","doi-asserted-by":"publisher","first-page":"982","DOI":"10.1128\/JB.01209-13","article-title":"Addition of Escherichia coli K-12\nGrowth Observation and Gene Essentiality Data to the EcoCyc Database","volume":"196","author":"Mackie","year":"2014","journal-title":"J. Bacteriol."},{"issue":"1","key":"2026081808124013900_cit16","doi-asserted-by":"publisher","first-page":"2006","DOI":"10.1038\/msb4100050","article-title":"Construction\nof Escherichia coli K-12 In-frame, Single-gene Knockout\nMutants: The Keio Collection","volume":"2","author":"Baba","year":"2006","journal-title":"Mol. Syst. Biol."},{"key":"2026081808124013900_cit17","doi-asserted-by":"publisher","first-page":"1091677","DOI":"10.3389\/fmolb.2023.1091677","article-title":"In Vivo Client Proteins of the Chaperonin GroEL-GroES\nProvide Insight into the Role of Chaperones in Protein Evolution","volume":"10","author":"Taguchi","year":"2023","journal-title":"Front. Mol. 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Unfolding of Phenylmethanesulfonyl.Alpha.-Chymotrypsin\nUsing Different Denaturants","volume":"27","author":"Santoro","year":"1988","journal-title":"Biochemistry"},{"issue":"10","key":"2026081808124013900_cit33","doi-asserted-by":"publisher","first-page":"1395","DOI":"10.3390\/biomedicines9101395","article-title":"Protein Unfolding:\nDenaturant vs. Force","volume":"9","author":"Kelly","year":"2021","journal-title":"Biomedicines"},{"issue":"7","key":"2026081808124013900_cit34","doi-asserted-by":"publisher","first-page":"1811","DOI":"10.1110\/ps.051387005","article-title":"Urea-induced Denaturation of Apolipoprotein\nSerum Amyloid\nA Reveals Marginal Stability of Hexamer","volume":"14","author":"Wang","year":"2005","journal-title":"Protein\nSci."},{"issue":"11","key":"2026081808124013900_cit35","doi-asserted-by":"publisher","first-page":"999","DOI":"10.1093\/protein\/11.11.999","article-title":"A Cooperative\nFolding Unit in HIV-1\nProtease. Implications for Protein Stability and Occurrence of Drug-Induced\nMutations","volume":"11","author":"Wallqvist","year":"1998","journal-title":"Protein Eng."},{"issue":"20","key":"2026081808124013900_cit36","doi-asserted-by":"publisher","first-page":"6861","DOI":"10.3390\/molecules27206861","article-title":"Conformational\nStability and Denaturation Processes\nof Proteins Investigated by Electrophoresis under Extreme Conditions","volume":"27","author":"Masson","year":"2022","journal-title":"Molecules"},{"issue":"7","key":"2026081808124013900_cit37","doi-asserted-by":"publisher","first-page":"2152","DOI":"10.1073\/pnas.86.7.2152","article-title":"Identifying Determinants\nof Folding and Activity for\na Protein of Unknown Structure","volume":"86","author":"Bowie","year":"1989","journal-title":"Proc. Natl.\nAcad. Sci. U. S. 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BioAllied Sci."},{"issue":"11","key":"2026081808124013900_cit39","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0050380","article-title":"Thermal, Chemical\nand pH Induced\nDenaturation of a Multimeric \u03b2-Galactosidase Reveals Multiple\nUnfolding Pathways","volume":"7","author":"Kishore","year":"2012","journal-title":"PLoS One"},{"issue":"12","key":"2026081808124013900_cit40","doi-asserted-by":"publisher","first-page":"22518","DOI":"10.3390\/ijms151222518","article-title":"Intrinsic Tryptophan\nFluorescence in the Detection and Analysis of\nProteins: A Focus on Fo\u0308rster Resonance Energy Transfer Techniques","volume":"15","author":"Ghisaidoobe","year":"2014","journal-title":"Int. J. Mol. Sci."},{"issue":"45","key":"2026081808124013900_cit41","doi-asserted-by":"publisher","first-page":"11738","DOI":"10.1021\/ja982019w","article-title":"Solution NMR Studies\nof a 42 KDa Escherichia coli Maltose Binding Protein\/\u03b2-Cyclodextrin\nComplex: Chemical Shift Assignments and Analysis","volume":"120","author":"Gardner","year":"1998","journal-title":"J. Am. Chem. Soc."},{"key":"2026081808124013900_cit42","doi-asserted-by":"publisher","first-page":"106733","DOI":"10.1016\/j.bpc.2021.106733","article-title":"Differential Effect of Polyol and Sugar Osmolytes on\nthe Refolding of Homologous Alpha Amylases: A Comparative Study","volume":"281","author":"Ahmad","year":"2022","journal-title":"Biophys. Chem."},{"issue":"10","key":"2026081808124013900_cit43","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0109408","article-title":"Structural\nCharacteristic of the Initial Unfolded State on Refolding Determines\nCatalytic Efficiency of the Folded Protein in Presence of Osmolytes","volume":"9","author":"Warepam","year":"2014","journal-title":"PLoS One"},{"issue":"1","key":"2026081808124013900_cit44","doi-asserted-by":"publisher","first-page":"90","DOI":"10.1016\/j.bpc.2011.05.012","article-title":"Osmolyte Effects on Protein Stability and Solubility:\nA Balancing Act between Backbone and Side-Chains","volume":"159","author":"Auton","year":"2011","journal-title":"Biophys. Chem."},{"issue":"4","key":"2026081808124013900_cit45","doi-asserted-by":"publisher","first-page":"211","DOI":"10.1080\/10409230600760382","article-title":"GroEL-Mediated\nProtein Folding: Making the Impossible,\nPossible","volume":"41","author":"Lin","year":"2006","journal-title":"Crit. Rev. Biochem. Mol. Biol."},{"issue":"10","key":"2026081808124013900_cit46","doi-asserted-by":"publisher","first-page":"2138","DOI":"10.1002\/pro.5560041020","article-title":"Denaturant m Values\nand Heat Capacity Changes: Relation to Changes in Accessible Surface\nAreas of Protein Unfolding","volume":"4","author":"Myers","year":"1995","journal-title":"Protein Sci."},{"issue":"10","key":"2026081808124013900_cit47","doi-asserted-by":"publisher","first-page":"6827","DOI":"10.1039\/D2RA07931C","article-title":"Ocular Protein Optineurin\nShows Reversibility from Unfolded States\nand Exhibits Chaperone-like Activity","volume":"13","author":"Dixit","year":"2023","journal-title":"RSC Adv."}],"container-title":["Biochemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.biochem.6c00531","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/bichaw\/article-pdf\/65\/16\/2522\/66468009\/acs.biochem.6c00531.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/bichaw\/article-pdf\/65\/16\/2522\/66468009\/acs.biochem.6c00531.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T12:37:52Z","timestamp":1787056672000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/bichaw\/article\/65\/16\/2522\/5250890\/Spontaneous-Refolding-of-Escherichia-coli"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,10]]},"references-count":47,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2026,8,10]]},"published-print":{"date-parts":[[2026,8,18]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.biochem.6c00531","relation":{},"ISSN":["0006-2960","1520-4995"],"issn-type":[{"value":"0006-2960","type":"print"},{"value":"1520-4995","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,10]]}}}