native gel electrophoresis was conducted as described . for crosslinking , 1 - 5 mm dss
rbc lysates were treated analogously but utilizing 1 mm bs . to purify syn from fresh or packed frozen rbc , an initial 25% ( nh4)2so4 cut followed by a 50% ( nh4)2so4 precipitation substantially enriched syn .
the resolubilized 50% pellet was injected onto a hydrophobic interaction column ( hitrap phenyl hp , ge healthcare ) and eluted in a 1 m to 0 m ( nh4)2so4 , ph 7 . alternatively , syn - overexpressing 3d5 neuroblastoma cell lysate after ( nh4)2so4 was injected onto a 5 ml hitrap q hp column . a 25 to 500 mm nacl ( ph 8.0 ) gradient eluted syn at 300 mm nacl .
syn from both cell sources underwent a final purification step on a superdex 75 sec column .
sedimentation equilibrium data were acquired on a beckman xl - i analytical ultracentrifuge at speeds of 12k , 16k , and 20k rpm ( an-60 ti rotor ) and protein concentrations of 0.6 , 1.1 and 1.6 mg / ml . cd spectroscopy for lipid - induced syn folding was conducted in the presence of 4 mm popc / pops ( 4:1 ) suvs .
spr spectroscopy was conducted as described . to quantify amyloid fibril growth , aliquots ( 10 l ) of purified syn
were added to a 10 m thioflavin t ( tht ) solution in 10 mm glycine buffer , ph 9 .
tht fluorescence was measured by exciting at 444 nm and scanning the emission wavelengths from 460 to 550 nm .
 parkinson disease ( pd ) is the second most common neurodegenerative disorder1,2 . 
 growing evidence suggests a causative role of misfolded forms of the protein , -synuclein ( syn ) , in the pathogenesis of pd3,4 . 
 intraneuronal aggregates of syn occur in lewy bodies and lewy neurites5 , the cytopathological hallmarks of pd and the related disorders called synucleinopathies . 
 syn has long been defined as a  natively unfolded  
 monomer of 14 kda6 that is believed to acquire -helical secondary structure only upon binding to lipid vesicles7 . 
 this concept derives from the widespread use of recombinant bacterial expression protocols for in vitro studies , and of overexpression , sample heating and/or denaturing gels for cell culture and tissue studies . 
 in contrast , we report that endogenous syn isolated and analyzed under non - denaturing conditions from neuronal and non - neuronal cell lines , brain tissue and living human cells occurs in large part as a folded tetramer of 58 kda . 
 multiple methods , including analytical ultracentrifugation , scanning transmission electron microscopy and in vivo cell crosslinking , confirmed the occurrence of the tetramer . 
 native , cell - derived syn showed -helical structure without lipid addition and had much greater lipid binding capacity than the recombinant syn studied heretofore . whereas recombinantly expressed monomers readily aggregated into amyloid - like fibrils in vitro , native human tetramers underwent little or no amyloid - like aggregation . 
 based on these findings , we propose that destabilization of the helically folded tetramer precedes syn misfolding and aggregation in pd and other human synucleinopathies and that small molecules which stabilize the physiological tetramer could reduce syn pathogenicity . 
Methods summary
Supplementary Material