000836076 001__ 836076
000836076 005__ 20210129230856.0
000836076 0247_ $$2doi$$a10.1371/journal.pone.0177024
000836076 0247_ $$2Handle$$a2128/15130
000836076 0247_ $$2WOS$$aWOS:000400648500123
000836076 037__ $$aFZJ-2017-05200
000836076 082__ $$a500
000836076 1001_ $$0P:(DE-HGF)0$$aKroeger, Tobias$$b0
000836076 245__ $$aEDTA aggregates induce SYPRO orange-based fluorescence in thermal shift assay
000836076 260__ $$aLawrence, Kan.$$bPLoS$$c2017
000836076 3367_ $$2DRIVER$$aarticle
000836076 3367_ $$2DataCite$$aOutput Types/Journal article
000836076 3367_ $$0PUB:(DE-HGF)16$$2PUB:(DE-HGF)$$aJournal Article$$bjournal$$mjournal$$s1502201472_1302
000836076 3367_ $$2BibTeX$$aARTICLE
000836076 3367_ $$2ORCID$$aJOURNAL_ARTICLE
000836076 3367_ $$00$$2EndNote$$aJournal Article
000836076 520__ $$aEthylenediaminetetraacetic acid (EDTA) is widely used in the life sciences as chelating ligand of metal ions. However, formation of supramolecular EDTA aggregates at pH > 8 has been reported, which may lead to artifactual assay results. When applied as a buffer component at pH ≈ 10 in differential scanning fluorimetry (TSA) using SYPRO Orange as fluorescent dye, we observed a sharp change in fluorescence intensity about 20°C lower than expected for the investigated protein. We hypothesized that this change results from SYPRO Orange/EDTA interactions. TSA experiments in the presence of SYPRO Orange using solutions that contain EDTA-Na+ but no protein were performed. The TSA experiments provide evidence that suggests that at pH > 9, EDTA4- interacts with SYPRO Orange in a temperature-dependent manner, leading to a fluorescence signal yielding a “denaturation temperature” of ~68°C. Titrating Ca2+ to SYPRO Orange and EDTA solutions quenched fluorescence. Ethylene glycol tetraacetic acid (EGTA) behaved similarly to EDTA. Analytical ultracentrifugation corroborated the formation of EDTA aggregates. Molecular dynamics simulations of free diffusion of EDTA-Na+ and SYPRO Orange of in total 27 μs suggested the first structural model of EDTA aggregates in which U-shaped EDTA4- arrange in an inverse bilayer-like manner, exposing ethylene moieties to the solvent, with which SYPRO Orange interacts. We conclude that EDTA aggregates induce a SYPRO Orange-based fluorescence in TSA. These results make it relevant to ascertain that future TSA results are not influenced by interference between EDTA, or EDTA-related molecules, and the fluorescent dye.
000836076 536__ $$0G:(DE-HGF)POF3-553$$a553 - Physical Basis of Diseases (POF3-553)$$cPOF3-553$$fPOF III$$x0
000836076 7001_ $$0P:(DE-Juel1)172887$$aFrieg, Benedikt$$b1
000836076 7001_ $$0P:(DE-HGF)0$$aZhang, Tao$$b2
000836076 7001_ $$0P:(DE-HGF)0$$aHansen, Finn K.$$b3
000836076 7001_ $$0P:(DE-HGF)0$$aMarmann, Andreas$$b4
000836076 7001_ $$0P:(DE-HGF)0$$aProksch, Peter$$b5
000836076 7001_ $$0P:(DE-Juel1)162443$$aNagel-Steger, Luitgard$$b6
000836076 7001_ $$0P:(DE-HGF)0$$aGroth, Georg$$b7
000836076 7001_ $$0P:(DE-HGF)0$$aSmits, Sander H. J.$$b8
000836076 7001_ $$0P:(DE-Juel1)172663$$aGohlke, Holger$$b9$$eCorresponding author
000836076 773__ $$0PERI:(DE-600)2267670-3$$a10.1371/journal.pone.0177024$$pe0177024$$tPLoS one$$v12$$x1932-6203$$y2017
000836076 8564_ $$uhttps://juser.fz-juelich.de/record/836076/files/EDTA%20aggregates%20induce%20SYPRO%20orange-based%20fluorescence%20in%20thermal%20shift%20assay.pdf$$yOpenAccess
000836076 909CO $$ooai:juser.fz-juelich.de:836076$$pdnbdelivery$$pVDB$$pdriver$$popen_access$$popenaire
000836076 915__ $$0StatID:(DE-HGF)0200$$2StatID$$aDBCoverage$$bSCOPUS
000836076 915__ $$0StatID:(DE-HGF)1050$$2StatID$$aDBCoverage$$bBIOSIS Previews
000836076 915__ $$0LIC:(DE-HGF)CCBY4$$2HGFVOC$$aCreative Commons Attribution CC BY 4.0
000836076 915__ $$0StatID:(DE-HGF)0600$$2StatID$$aDBCoverage$$bEbsco Academic Search
000836076 915__ $$0StatID:(DE-HGF)1040$$2StatID$$aDBCoverage$$bZoological Record
000836076 915__ $$0StatID:(DE-HGF)0100$$2StatID$$aJCR$$bPLOS ONE : 2015
000836076 915__ $$0StatID:(DE-HGF)0501$$2StatID$$aDBCoverage$$bDOAJ Seal
000836076 915__ $$0StatID:(DE-HGF)0500$$2StatID$$aDBCoverage$$bDOAJ
000836076 915__ $$0StatID:(DE-HGF)0111$$2StatID$$aWoS$$bScience Citation Index Expanded
000836076 915__ $$0StatID:(DE-HGF)0150$$2StatID$$aDBCoverage$$bWeb of Science Core Collection
000836076 915__ $$0StatID:(DE-HGF)9900$$2StatID$$aIF < 5
000836076 915__ $$0StatID:(DE-HGF)0510$$2StatID$$aOpenAccess
000836076 915__ $$0StatID:(DE-HGF)0030$$2StatID$$aPeer Review$$bASC
000836076 915__ $$0StatID:(DE-HGF)0310$$2StatID$$aDBCoverage$$bNCBI Molecular Biology Database
000836076 915__ $$0StatID:(DE-HGF)0300$$2StatID$$aDBCoverage$$bMedline
000836076 915__ $$0StatID:(DE-HGF)0199$$2StatID$$aDBCoverage$$bThomson Reuters Master Journal List
000836076 9141_ $$y2017
000836076 9101_ $$0I:(DE-588b)5008462-8$$6P:(DE-Juel1)172887$$aForschungszentrum Jülich$$b1$$kFZJ
000836076 9101_ $$0I:(DE-588b)5008462-8$$6P:(DE-HGF)0$$aForschungszentrum Jülich$$b2$$kFZJ
000836076 9101_ $$0I:(DE-588b)5008462-8$$6P:(DE-Juel1)162443$$aForschungszentrum Jülich$$b6$$kFZJ
000836076 9101_ $$0I:(DE-588b)5008462-8$$6P:(DE-Juel1)172663$$aForschungszentrum Jülich$$b9$$kFZJ
000836076 9131_ $$0G:(DE-HGF)POF3-553$$1G:(DE-HGF)POF3-550$$2G:(DE-HGF)POF3-500$$3G:(DE-HGF)POF3$$4G:(DE-HGF)POF$$aDE-HGF$$bKey Technologies$$lBioSoft – Fundamentals for future Technologies in the fields of Soft Matter and Life Sciences$$vPhysical Basis of Diseases$$x0
000836076 920__ $$lyes
000836076 9201_ $$0I:(DE-Juel1)ICS-6-20110106$$kICS-6$$lStrukturbiochemie$$x0
000836076 9801_ $$aFullTexts
000836076 980__ $$ajournal
000836076 980__ $$aVDB
000836076 980__ $$aUNRESTRICTED
000836076 980__ $$aI:(DE-Juel1)ICS-6-20110106
000836076 981__ $$aI:(DE-Juel1)IBI-7-20200312