{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T02:39:33Z","timestamp":1791167973175,"version":"4.1.0"},"reference-count":46,"publisher":"Wiley","issue":"7","license":[{"start":{"date-parts":[[2009,4,13]],"date-time":"2009-04-13T00:00:00Z","timestamp":1239580800000},"content-version":"vor","delay-in-days":2843,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Protein Science"],"published-print":{"date-parts":[[2001,7]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>The DNA repair protein XPA recognizes a wide variety of bulky lesions and interacts with several other proteins during nucleotide excision repair. We recently identified regions of intrinsic order and disorder in full length<jats:italic>Xenopus<\/jats:italic>XPA (xXPA) protein using an experimental approach that combined time\u2010resolved trypsin proteolysis and electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance (ESI\u2010FTICR) mass spectrometry (MS). MS data were consistent with the interpretation that xXPA contains no post\u2010translational modifications. Here we characterize the discrepancy between the calculated molecular weight (31 kDa) for xXPA and its apparent molecular weight on SDS\u2010PAGE (multiple bands from \u223c40\u201345 kDa) and gel filtration chromatography (\u223c92 kDa), as well as the consequences of DNA binding on its anomalous mobility. Iodoacetamide treatment of xXPA prior to SDS\u2010PAGE yielded a single 42\u2010kDa band, showing that covalent modification of Cys did not correct aberrant mobility. Determination of sulfhydryl content in xXPA with Ellman's reagent revealed that all nine Cys in active protein are reduced. Unexpectedly, structural constraints induced by intramolecular glutaraldehyde crosslinks in xXPA produced a \u223c32\u2010kDa monomer in closer agreement with its calculated molecular weight. To investigate whether binding to DNA alters xXPA's anomalous migration, we used gel filtration chromatography. For the first time, we purified stable complexes of xXPA and DNA \u00b1 cisplatin \u00b1 mismatches. xXPA showed at least 10\u2010fold higher affinity for cisplatin DNA \u00b1 mismatches compared to undamaged DNA \u00b1 mismatches. In all cases, DNA binding did not correct xXPA's anomalous migration. To test predictions that a Glu\u2010rich region (EEEEAEE) and\/or disordered N\u2010 and C\u2010terminal domains were responsible for xXPA's aberrant mobility, the molecular weights of partial proteolytic fragments from \u223c5 to 25 kDa separated by reverse\u2010phase HPLC and precisely determined by ESI\u2010FTICR MS were correlated with their migration on SDS\u2010PAGE. Every partial tryptic fragment analyzed within this size range exhibited 10%\u201350% larger molecular weights than expected. Thus, both the disordered domains and the Glu\u2010rich region in xXPA are primarily responsible for the aberrant mobility phenomena.<\/jats:p>","DOI":"10.1110\/ps.ps.40101","type":"journal-article","created":{"date-parts":[[2010,10,1]],"date-time":"2010-10-01T14:09:28Z","timestamp":1285942168000},"page":"1353-1362","source":"Crossref","is-referenced-by-count":54,"title":["Aberrant mobility phenomena of the DNA repair protein XPA"],"prefix":"10.1002","volume":"10","author":[{"given":"Lilia M.","family":"Iakoucheva","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amy L.","family":"Kimzey","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christophe D.","family":"Masselon","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard D.","family":"Smith","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"A. 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