{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T18:15:44Z","timestamp":1791224144897,"version":"4.1.0"},"reference-count":50,"publisher":"American Chemical Society (ACS)","issue":"2","license":[{"start":{"date-parts":[[2015,12,9]],"date-time":"2015-12-09T00:00:00Z","timestamp":1449619200000},"content-version":"vor","delay-in-days":365,"URL":"https:\/\/pubs.acs.org\/page\/policy\/authorchoice_termsofuse.html"}],"funder":[{"DOI":"10.13039\/100000065","name":"National Institute of Neurological Disorders and Stroke","doi-asserted-by":"publisher","award":["1 R01 NS083845"],"award-info":[{"award-number":["1 R01 NS083845"]}],"id":[{"id":"10.13039\/100000065","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2015,1,20]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Despite two decades of research, the structure\u2013function relationships of endogenous, physiological forms of \u03b1-synuclein (\u03b1Syn) are not well understood. Most in vitro studies of this Parkinson\u2019s disease-related protein have focused on recombinant \u03b1Syn that is unfolded and monomeric, assuming that this represents its state in the normal human brain. Recently, we have provided evidence that \u03b1Syn exists in considerable part in neurons, erythrocytes, and other cells as a metastable multimer that principally sizes as a tetramer. In contrast to recombinant \u03b1Syn, physiological tetramers purified from human erythrocytes have substantial \u03b1-helical content and resist pathological aggregation into \u03b2-sheet rich fibers. Here, we report the first method to fully purify soluble \u03b1Syn from the most relevant source, human brain. We describe protocols that purify \u03b1Syn to homogeneity from nondiseased human cortex using ammonium sulfate precipitation, gel filtration, and ion exchange, hydrophobic interaction, and affinity chromatographies. Cross-linking of the starting material and the partially purified chromatographic fractions revealed abundant \u03b1Syn multimers, including apparent tetramers, but these were destabilized in large part to monomers during the final purification step. The method also fully purified the homologue \u03b2-synuclein, with a similar outcome. Circular dichroism spectroscopy showed that purified, brain-derived \u03b1Syn can display more helical content than the recombinant protein, but this result varied. Collectively, our data suggest that purifying \u03b1Syn to homogeneity destabilizes native, \u03b1-helix-rich multimers that exist in intact and partially purified brain samples. This finding suggests existence of a stabilizing cofactor (e.g., a small lipid) present inside neurons that is lost during final purification.<\/jats:p>","DOI":"10.1021\/bi501188a","type":"journal-article","created":{"date-parts":[[2014,12,9]],"date-time":"2014-12-09T17:37:07Z","timestamp":1418146627000},"page":"279-292","source":"Crossref","is-referenced-by-count":74,"title":["Purification of \u03b1-Synuclein from Human\nBrain Reveals an Instability of Endogenous Multimers as the Protein\nApproaches Purity"],"prefix":"10.1021","volume":"54","author":[{"given":"Eric S.","family":"Luth","sequence":"first","affiliation":[{"name":"Brigham and Women\u2019s Hospital and Harvard Medical School , , , ,","place":["Boston, Massachusetts, United States, 02115"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tim","family":"Bartels","sequence":"additional","affiliation":[{"name":"Brigham and Women\u2019s Hospital and Harvard Medical School , , , ,","place":["Boston, Massachusetts, United States, 02115"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ulf","family":"Dettmer","sequence":"additional","affiliation":[{"name":"Brigham and Women\u2019s Hospital and Harvard Medical School , , , ,","place":["Boston, Massachusetts, United States, 02115"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nora C.","family":"Kim","sequence":"additional","affiliation":[{"name":"Brigham and Women\u2019s Hospital and Harvard Medical School , , , ,","place":["Boston, Massachusetts, United States, 02115"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dennis J.","family":"Selkoe","sequence":"additional","affiliation":[{"name":"Brigham and Women\u2019s Hospital and Harvard Medical School , , , ,","place":["Boston, Massachusetts, United States, 02115"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2014,12,9]]},"reference":[{"key":"2026090205335211900_cit1","doi-asserted-by":"crossref","first-page":"2045","DOI":"10.1126\/science.276.5321.2045","article-title":"Mutation\nin the alpha-synuclein gene identified in families with Parkinson\u2019s\ndisease","volume":"276","author":"Polymeropoulos","year":"1997","journal-title":"Science"},{"key":"2026090205335211900_cit2","doi-asserted-by":"crossref","first-page":"1167","DOI":"10.1016\/S0140-6736(04)17103-1","article-title":"Alpha-synuclein locus duplication\nas a cause of familial Parkinson\u2019s disease","volume":"364","author":"Chartier-Harlin","year":"2004","journal-title":"Lancet"},{"key":"2026090205335211900_cit3","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1016\/j.neulet.2013.04.058","article-title":"\u03b1-Synuclein\nmutations cluster\naround a putative protein loop","volume":"546","author":"Kara","year":"2013","journal-title":"Neurosci. Lett."},{"key":"2026090205335211900_cit4","doi-asserted-by":"crossref","first-page":"841","DOI":"10.1126\/science.1090278","article-title":"alpha-Synuclein\nlocus triplication\ncauses Parkinson\u2019s disease","volume":"302","author":"Singleton","year":"2003","journal-title":"Science"},{"key":"2026090205335211900_cit5","doi-asserted-by":"crossref","first-page":"839","DOI":"10.1038\/42166","article-title":"Alpha-synuclein in Lewy bodies","volume":"388","author":"Spillantini","year":"1997","journal-title":"Nature"},{"key":"2026090205335211900_cit6","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1016\/j.bbadis.2011.10.002","article-title":"A-synuclein misfolding and Parkinson\u2019s\ndisease","volume":"1822","author":"Breydo","year":"2012","journal-title":"Biochim. Biophys. Acta"},{"key":"2026090205335211900_cit7","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1016\/j.expneurol.2007.05.022","article-title":"Cell systems\nand the toxic mechanism(s) of alpha-synuclein","volume":"209","author":"Cookson","year":"2008","journal-title":"Exp. Neurol."},{"key":"2026090205335211900_cit8","doi-asserted-by":"crossref","first-page":"7871","DOI":"10.1021\/bi030086j","article-title":"Zeroing in on the pathogenic form of alpha-synuclein\nand its mechanism of neurotoxicity in Parkinson\u2019s disease","volume":"42","author":"Volles","year":"2003","journal-title":"Biochemistry"},{"key":"2026090205335211900_cit9","doi-asserted-by":"crossref","first-page":"361","DOI":"10.1016\/0896-6273(95)90040-3","article-title":"Characterization of a novel protein\nregulated during the critical period for song learning in the zebra\nfinch","volume":"15","author":"George","year":"1995","journal-title":"Neuron"},{"key":"2026090205335211900_cit10","doi-asserted-by":"crossref","first-page":"467","DOI":"10.1016\/0896-6273(95)90302-X","article-title":"The precursor protein\nof non-A beta component of Alzheimer\u2019s disease amyloid is a\npresynaptic protein of the central nervous system","volume":"14","author":"Iwai","year":"1995","journal-title":"Neuron"},{"key":"2026090205335211900_cit11","doi-asserted-by":"crossref","first-page":"6371","DOI":"10.1074\/jbc.M112.403311","article-title":"In vivo cross-linking\nreveals principally oligomeric forms of \u03b1-synuclein and \u03b2-synuclein\nin neurons and non-neural cells","volume":"288","author":"Dettmer","year":"2013","journal-title":"J. Biol. Chem."},{"key":"2026090205335211900_cit12","doi-asserted-by":"crossref","first-page":"6365","DOI":"10.1523\/JNEUROSCI.20-17-06365.2000","article-title":"Subcellular localization of wild-type and Parkinson\u2019s disease-associated\nmutant alpha -synuclein in human and transgenic mouse brain","volume":"20","author":"Kahle","year":"2000","journal-title":"J. Neurosci."},{"key":"2026090205335211900_cit13","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1016\/S0896-6273(00)80886-7","article-title":"Mice lacking alpha-synuclein display\nfunctional deficits in the nigrostriatal dopamine system","volume":"25","author":"Abeliovich","year":"2000","journal-title":"Neuron"},{"key":"2026090205335211900_cit14","doi-asserted-by":"crossref","first-page":"11165","DOI":"10.1523\/JNEUROSCI.2559-04.2004","article-title":"Role of alpha-synuclein\nin presynaptic dopamine recruitment","volume":"24","author":"Yavich","year":"2004","journal-title":"J. Neurosci."},{"key":"2026090205335211900_cit15","doi-asserted-by":"crossref","first-page":"724","DOI":"10.1111\/j.1471-4159.2006.04098.x","article-title":"Abnormal compartmentalization\nof norepinephrine in mouse dentate gyrus in alpha-synuclein knockout\nand A30P transgenic mice","volume":"99","author":"Yavich","year":"2006","journal-title":"J. Neurochem."},{"key":"2026090205335211900_cit16","doi-asserted-by":"crossref","first-page":"11915","DOI":"10.1523\/JNEUROSCI.3821-06.2006","article-title":"Alpha-synuclein overexpression\nin PC12 and chromaffin cells impairs catecholamine release by interfering\nwith a late step in exocytosis","volume":"26","author":"Larsen","year":"2006","journal-title":"J. Neurosci."},{"key":"2026090205335211900_cit17","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1016\/j.neuron.2009.12.023","article-title":"Increased expression\nof alpha-synuclein reduces neurotransmitter release by inhibiting\nsynaptic vesicle reclustering after endocytosis","volume":"65","author":"Nemani","year":"2010","journal-title":"Neuron"},{"key":"2026090205335211900_cit18","doi-asserted-by":"crossref","first-page":"2605","DOI":"10.1523\/JNEUROSCI.2898-12.2013","article-title":"Mapping the subcellular\ndistribution of \u03b1-synuclein\nin neurons using genetically encoded probes for correlated light and\nelectron microscopy: implications for Parkinson\u2019s disease pathogenesis","volume":"33","author":"Boassa","year":"2013","journal-title":"J. Neurosci."},{"key":"2026090205335211900_cit19","doi-asserted-by":"crossref","first-page":"8083","DOI":"10.1523\/JNEUROSCI.1091-10.2010","article-title":"A pathologic cascade leading to synaptic dysfunction in alpha-synuclein-induced\nneurodegeneration","volume":"30","author":"Scott","year":"2010","journal-title":"J. Neurosci."},{"key":"2026090205335211900_cit20","doi-asserted-by":"crossref","first-page":"3571","DOI":"10.1038\/emboj.2010.223","article-title":"Inhibition of mitochondrial fusion\nby \u03b1-synuclein is rescued\nby PINK1, Parkin and DJ-1","volume":"29","author":"Kamp","year":"2010","journal-title":"EMBO J."},{"key":"2026090205335211900_cit21","doi-asserted-by":"crossref","first-page":"32486","DOI":"10.1074\/jbc.M110.139576","article-title":"Membrane curvature\ninduction and\ntubulation are common features of synucleins and apolipoproteins","volume":"285","author":"Varkey","year":"2010","journal-title":"J. Biol. Chem."},{"key":"2026090205335211900_cit22","doi-asserted-by":"crossref","first-page":"1829","DOI":"10.1074\/jbc.M112.418871","article-title":"Monomeric synucleins generate membrane curvature","volume":"288","author":"Westphal","year":"2013","journal-title":"J. Biol. Chem."},{"key":"2026090205335211900_cit23","doi-asserted-by":"crossref","first-page":"1663","DOI":"10.1126\/science.1195227","article-title":"Alpha-synuclein promotes SNARE-complex assembly in\nvivo and in vitro","volume":"329","author":"Burre\u0301","year":"2010","journal-title":"Science"},{"key":"2026090205335211900_cit24","doi-asserted-by":"crossref","first-page":"2385","DOI":"10.1021\/bi4002369","article-title":"\u03b1-Synuclein Can Inhibit SNARE-Mediated\nVesicle\nFusion through Direct Interactions with Lipid Bilayers","volume":"52","author":"DeWitt","year":"2013","journal-title":"Biochemistry"},{"key":"2026090205335211900_cit25","doi-asserted-by":"crossref","first-page":"383","DOI":"10.1016\/j.cell.2005.09.028","article-title":"Alpha-synuclein cooperates with CSPalpha\nin preventing neurodegeneration","volume":"123","author":"Chandra","year":"2005","journal-title":"Cell"},{"key":"2026090205335211900_cit26","doi-asserted-by":"crossref","first-page":"e00592","DOI":"10.7554\/eLife.00592","article-title":"Native \u03b1-synuclein\ninduces clustering of synaptic-vesicle mimics via binding to phospholipids\nand synaptobrevin-2\/VAMP2","volume":"2","author":"Diao","year":"2013","journal-title":"eLife"},{"key":"2026090205335211900_cit27","doi-asserted-by":"crossref","first-page":"13709","DOI":"10.1021\/bi961799n","article-title":"NACP, a\nprotein implicated in Alzheimer\u2019s disease and learning, is\nnatively unfolded","volume":"35","author":"Weinreb","year":"1996","journal-title":"Biochemistry"},{"key":"2026090205335211900_cit28","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1016\/S1016-8478(23)13265-1","article-title":"Evidence that\nthe precursor protein of non-A beta component of Alzheimer\u2019s\ndisease amyloid (NACP) has an extended structure primarily composed\nof random-coil","volume":"7","author":"Kim","year":"1997","journal-title":"Mol. Cells"},{"key":"2026090205335211900_cit29","doi-asserted-by":"crossref","first-page":"950","DOI":"10.1042\/BST20120096","article-title":"Bacterial in-cell NMR of human \u03b1-synuclein:\na disordered monomer by nature?","volume":"40","author":"Binolfi","year":"2012","journal-title":"Biochem. Soc.\nTrans."},{"key":"2026090205335211900_cit30","doi-asserted-by":"crossref","first-page":"e72286","DOI":"10.1371\/journal.pone.0072286","article-title":"In-cell NMR characterization of the\nsecondary structure populations of a disordered conformation of \u03b1-synuclein\nwithin E. coli cells","volume":"8","author":"Waudby","year":"2013","journal-title":"PLoS One"},{"key":"2026090205335211900_cit31","doi-asserted-by":"crossref","first-page":"19536","DOI":"10.1021\/ja208657z","article-title":"Explaining the structural plasticity\nof \u03b1-synuclein","volume":"133","author":"Ullman","year":"2011","journal-title":"J. Am. Chem. Soc."},{"key":"2026090205335211900_cit32","doi-asserted-by":"crossref","first-page":"3865","DOI":"10.1021\/ja310518p","article-title":"The dynamic structure\nof \u03b1-synuclein multimers","volume":"135","author":"Gurry","year":"2013","journal-title":"J. Am. Chem.\nSoc."},{"key":"2026090205335211900_cit33","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1038\/nature10324","article-title":"\u03b1-Synuclein\noccurs physiologically\nas a helically folded tetramer that resists aggregation","volume":"477","author":"Bartels","year":"2011","journal-title":"Nature"},{"key":"2026090205335211900_cit34","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1159\/000112832","article-title":"Red blood cells\nare the major source of alpha-synuclein in blood","volume":"5","author":"Barbour","year":"2008","journal-title":"Neurodegener. Dis."},{"key":"2026090205335211900_cit35","doi-asserted-by":"crossref","first-page":"e81314","DOI":"10.1371\/journal.pone.0081314","article-title":"A new method for quantitative immunoblotting\nof endogenous \u03b1-synuclein","volume":"8","author":"Newman","year":"2013","journal-title":"PLoS One"},{"key":"2026090205335211900_cit36","doi-asserted-by":"crossref","first-page":"E4\u20136\u2013discussion E6\u20137","DOI":"10.1038\/nature12125","article-title":"Properties of native brain \u03b1-synuclein","volume":"498","author":"Burre\u0301","year":"2013","journal-title":"Nature"},{"key":"2026090205335211900_cit37","doi-asserted-by":"crossref","first-page":"1463","DOI":"10.1002\/bip.360311304","article-title":"Parameters\nof helix-coil transition theory for alanine-based peptides of varying\nchain lengths in water","volume":"31","author":"Scholtz","year":"1991","journal-title":"Biopolymers"},{"key":"2026090205335211900_cit38","doi-asserted-by":"crossref","first-page":"850","DOI":"10.1021\/ac950914h","article-title":"Mass spectrometric\nsequencing of proteins silver-stained polyacrylamide gels","volume":"68","author":"Shevchenko","year":"1996","journal-title":"Anal. Chem."},{"key":"2026090205335211900_cit39","doi-asserted-by":"crossref","first-page":"D191","DOI":"10.1093\/nar\/gku469","article-title":"Activities at\nthe Universal Protein Resource (UniProt)","volume":"42","author":"UniProt Consortium.","year":"2014","journal-title":"Nucleic\nAcids Res."},{"key":"2026090205335211900_cit40","doi-asserted-by":"crossref","first-page":"2050","DOI":"10.1096\/fj.05-5422com","article-title":"Detection\nof novel intracellular alpha-synuclein oligomeric species by fluorescence\nlifetime imaging","volume":"20","author":"Klucken","year":"2006","journal-title":"FASEB J."},{"key":"2026090205335211900_cit41","doi-asserted-by":"crossref","first-page":"17797","DOI":"10.1073\/pnas.1113260108","article-title":"A soluble \u03b1-synuclein construct forms a dynamic\ntetramer","volume":"108","author":"Wang","year":"2011","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"2026090205335211900_cit42","doi-asserted-by":"crossref","first-page":"601","DOI":"10.1002\/pro.2056","article-title":"N-Terminal acetylation\nis critical for forming \u03b1-helical\noligomer of \u03b1-synuclein","volume":"21","author":"Trexler","year":"2012","journal-title":"Protein Sci."},{"key":"2026090205335211900_cit43","doi-asserted-by":"crossref","first-page":"e103727","DOI":"10.1371\/journal.pone.0103727","article-title":"N-Alpha-acetylation\nof \u03b1-synuclein increases its helical folding propensity, GM1\nbinding specificity and resistance to aggregation","volume":"9","author":"Bartels","year":"2014","journal-title":"PLoS One"},{"key":"2026090205335211900_cit44","doi-asserted-by":"crossref","first-page":"9443","DOI":"10.1074\/jbc.273.16.9443","article-title":"Stabilization of\nalpha-synuclein secondary structure upon binding to synthetic membranes","volume":"273","author":"Davidson","year":"1998","journal-title":"J. Biol. Chem."},{"key":"2026090205335211900_cit45","doi-asserted-by":"crossref","first-page":"7929","DOI":"10.1074\/jbc.C113.538249","article-title":"Evidence of\nNative \u03b1-Synuclein Conformers in the Human Brain","volume":"14","author":"Gould","year":"2014","journal-title":"J. Biol. Chem."},{"key":"2026090205335211900_cit46","doi-asserted-by":"crossref","first-page":"1015","DOI":"10.1016\/j.jmb.2004.05.068","article-title":"Expression, purification and the 1.8 angstroms resolution\ncrystal structure of human neuron specific enolase","volume":"341","author":"Chai","year":"2004","journal-title":"J. Mol. Biol."},{"key":"2026090205335211900_cit47","doi-asserted-by":"crossref","first-page":"29739","DOI":"10.1074\/jbc.M600933200","article-title":"Phosphorylation of Ser-129 is the\ndominant pathological modification of alpha-synuclein in familial\nand sporadic Lewy body disease","volume":"281","author":"Anderson","year":"2006","journal-title":"J. Biol. Chem."},{"key":"2026090205335211900_cit48","doi-asserted-by":"crossref","first-page":"2319","DOI":"10.1016\/j.cub.2014.08.027","article-title":"\u03b1-Synuclein\nMultimers Cluster Synaptic Vesicles and Attenuate Recycling.","volume":"24","author":"Wang","year":"2014","journal-title":"Curr. Biol."},{"key":"2026090205335211900_cit49","doi-asserted-by":"crossref","first-page":"15345","DOI":"10.1074\/jbc.M111.318949","article-title":"\u03b1-Synuclein\nin central nervous system and from erythrocytes, mammalian cells,\nand Escherichia coli exists predominantly as disordered\nmonomer","volume":"287","author":"Fauvet","year":"2012","journal-title":"J. Biol. Chem."},{"key":"2026090205335211900_cit50","doi-asserted-by":"crossref","first-page":"1231","DOI":"10.1016\/j.bbapap.2010.01.017","article-title":"Understanding protein\nnon-folding","volume":"1804","author":"Uversky","year":"2010","journal-title":"Biochim. Biophys. Acta"}],"container-title":["Biochemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/bi501188a","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/bichaw\/article-pdf\/54\/2\/279\/15709798\/bi501188a.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/bichaw\/article-pdf\/54\/2\/279\/15709798\/bi501188a.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T09:34:03Z","timestamp":1788341643000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/bichaw\/article\/54\/2\/279\/1478048\/Purification-of-Synuclein-from-Human-Brain-Reveals"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2014,12,9]]},"references-count":50,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2014,12,9]]},"published-print":{"date-parts":[[2015,1,20]]}},"URL":"https:\/\/doi.org\/10.1021\/bi501188a","relation":{},"ISSN":["0006-2960","1520-4995"],"issn-type":[{"value":"0006-2960","type":"print"},{"value":"1520-4995","type":"electronic"}],"subject":[],"published":{"date-parts":[[2014,12,9]]}}}