{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T11:02:44Z","timestamp":1768474964116,"version":"3.49.0"},"reference-count":61,"publisher":"Wiley","issue":"6","license":[{"start":{"date-parts":[[2014,6,19]],"date-time":"2014-06-19T00:00:00Z","timestamp":1403136000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["R01 GM092999"],"award-info":[{"award-number":["R01 GM092999"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000049","name":"National Institute on Aging","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100000049","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Journal of Neurochemistry"],"published-print":{"date-parts":[[2014,9]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>The oxidation of a key cysteine residue (Cys106) in the parkinsonism\u2010associated protein <jats:styled-content style=\"fixed-case\">DJ<\/jats:styled-content>\u20101 regulates its ability to protect against oxidative stress and mitochondrial damage. Cys106 interacts with a neighboring protonated Glu18 residue, stabilizing the Cys106\u2010<jats:styled-content style=\"fixed-case\">SO<\/jats:styled-content><jats:sub>2<\/jats:sub><jats:sup>\u2212<\/jats:sup> (sulfinic acid) form of <jats:styled-content style=\"fixed-case\">DJ<\/jats:styled-content>\u20101. To study this important post\u2010translational modification, we previously designed several Glu18 mutations (E18N, E18D, E18Q) that alter the oxidative propensity of Cys106. However, recent results suggest these Glu18 mutations cause loss of <jats:styled-content style=\"fixed-case\">DJ<\/jats:styled-content>\u20101 dimerization, which would severely compromise the protein's function. The purpose of this study was to conclusively determine the oligomerization state of these mutants using X\u2010ray crystallography, <jats:styled-content style=\"fixed-case\">NMR<\/jats:styled-content> spectroscopy, thermal stability analysis, circular dichroism spectroscopy, sedimentation equilibrium ultracentrifugation, and cross\u2010linking. We found that all of the Glu18 <jats:styled-content style=\"fixed-case\">DJ<\/jats:styled-content>\u20101 mutants were dimeric. Thiol cross\u2010linking indicates that these mutant dimers are more flexible than the wild\u2010type protein and can form multiple cross\u2010linked dimeric species due to the transient exposure of cysteine residues that are inaccessible in the wild\u2010type protein. The enhanced flexibility of Glu18 <jats:styled-content style=\"fixed-case\">DJ<\/jats:styled-content>\u20101 mutants provides a parsimonious explanation for their lower observed cross\u2010linking efficiency in cells. In addition, thiol cross\u2010linkers may have an underappreciated value as qualitative probes of protein conformational flexibility.<jats:boxed-text content-type=\"graphic\" position=\"anchor\"><jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" mimetype=\"image\/png\" position=\"anchor\" specific-use=\"enlarged-web-image\" xlink:href=\"graphic\/jnc12763-fig-0009-m.png\"><jats:alt-text>image<\/jats:alt-text><\/jats:graphic><\/jats:boxed-text>\n<\/jats:p><jats:p>DJ\u20101 is a homodimeric protein that protects cells against oxidative stress. Designed mutations that influence the regulatory oxidation of a key cysteine residue have recently been proposed to disrupt DJ\u20101 dimerization. We use cysteine cross\u2010linking and various biophysical techniques to show that these DJ\u20101 mutants form dimers with increased conformational flexibility.<\/jats:p>","DOI":"10.1111\/jnc.12763","type":"journal-article","created":{"date-parts":[[2014,5,16]],"date-time":"2014-05-16T07:17:49Z","timestamp":1400224669000},"page":"839-853","source":"Crossref","is-referenced-by-count":16,"title":["Use of cysteine\u2010reactive cross\u2010linkers to probe conformational flexibility of human <scp>DJ<\/scp>\u20101 demonstrates that Glu18 mutations are dimers"],"prefix":"10.1111","volume":"130","author":[{"given":"Janani","family":"Prahlad","sequence":"first","affiliation":[{"name":"Department of Biochemistry and the Redox Biology Center University of Nebraska Lincoln Nebraska USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David N.","family":"Hauser","sequence":"additional","affiliation":[{"name":"Cell Biology and Gene Expression Section Laboratory of Neurogenetics National Institute on Aging Bethesda Maryland USA"},{"name":"Department of Neuroscience Brown University\/National Institutes of Health Graduate Partnership Program Brown University Providence Rhode Island USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicole M.","family":"Milkovic","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and the Redox Biology Center University of Nebraska Lincoln Nebraska USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mark R.","family":"Cookson","sequence":"additional","affiliation":[{"name":"Cell Biology and Gene Expression Section Laboratory of Neurogenetics National Institute on Aging Bethesda Maryland USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mark A.","family":"Wilson","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and the Redox Biology Center University of Nebraska Lincoln Nebraska USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2014,6,19]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444912001308"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0709379104"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1303444110"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M806599200"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1077209"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0402959101"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1089\/ars.2013.5446"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1093\/hmg\/ddq167"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF00197809"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444904019158"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0021889803006721"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0040501"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1110\/ps.51201"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature09536"},{"key":"e_1_2_8_16_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0911175107"},{"key":"e_1_2_8_17_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.nbd.2012.10.011"},{"key":"e_1_2_8_18_1","doi-asserted-by":"publisher","DOI":"10.1002\/(SICI)1097-4644(19990301)72:3<435::AID-JCB12>3.0.CO;2-H"},{"key":"e_1_2_8_19_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444903021784"},{"key":"e_1_2_8_20_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M305878200"},{"key":"e_1_2_8_21_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0014-5793(03)00764-6"},{"key":"e_1_2_8_22_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi7001778"},{"key":"e_1_2_8_23_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.neures.2010.04.002"},{"key":"e_1_2_8_24_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.conb.2011.10.016"},{"key":"e_1_2_8_25_1","doi-asserted-by":"publisher","DOI":"10.1093\/hmg\/ddq288"},{"key":"e_1_2_8_26_1","first-page":"313","article-title":"Using NMRView to visualize and analyze the NMR spectra of macromolecules","volume":"278","author":"Johnson B. 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