α-Synuclein (αSyn) is naturally present as a helically folded tetramer in cells, resisting aggregation. This study challenges the long-held belief that αSyn is a natively unfolded monomer. Using non-denaturing methods, researchers found that αSyn exists predominantly as a ~58 kDa tetramer in neuronal and non-neuronal cells, brain tissue, and human red blood cells. Multiple techniques, including analytical ultracentrifugation, scanning transmission electron microscopy, and in vivo crosslinking, confirmed the tetrameric structure. Native αSyn showed α-helical structure without lipid addition and had greater lipid binding capacity than recombinant αSyn. Recombinant monomers readily aggregated into amyloid-like fibrils, while native tetramers showed little aggregation. These findings suggest that destabilization of the helically folded tetramer precedes αSyn misfolding and aggregation in Parkinson's disease and other synucleinopathies. Stabilizing the physiological tetramer could reduce αSyn pathogenicity. The study used native gel electrophoresis, Clear Native PAGE, and in vivo crosslinking to analyze αSyn in cells. αSyn was detected as a ~45-50 kDa species in various cell lines and in the frontal cortex of wild-type mice. In human red blood cells, αSyn migrated at ~55-60 kDa on Clear Native PAGE, consistent with a tetramer. Scanning transmission electron microscopy and sedimentation equilibrium analytical ultracentrifugation further supported the tetrameric structure. Circular dichroism spectroscopy showed α-helical structure in native tetramers, while recombinant monomers underwent a random coil to α-helix transition upon lipid interaction. Phospholipid analysis and mass spectrometry confirmed the absence of significant lipid association in native tetramers. The study also compared αSyn from different cell types and purification methods, finding consistent tetrameric structures. Surface plasmon resonance showed increased lipid binding by native tetramers compared to recombinant monomers. Thioflavin T fluorescence assays revealed no fibril formation by native tetramers, suggesting they are aggregation-resistant. These findings indicate that the predominant physiological form of αSyn is a helically folded tetramer, which is less prone to aggregation than monomers. The study highlights the importance of non-denaturing methods in accurately determining the native state of αSyn and suggests that stabilizing the tetramer could be a therapeutic approach for Parkinson's disease and other synucleinopathies.α-Synuclein (αSyn) is naturally present as a helically folded tetramer in cells, resisting aggregation. This study challenges the long-held belief that αSyn is a natively unfolded monomer. Using non-denaturing methods, researchers found that αSyn exists predominantly as a ~58 kDa tetramer in neuronal and non-neuronal cells, brain tissue, and human red blood cells. Multiple techniques, including analytical ultracentrifugation, scanning transmission electron microscopy, and in vivo crosslinking, confirmed the tetrameric structure. Native αSyn showed α-helical structure without lipid addition and had greater lipid binding capacity than recombinant αSyn. Recombinant monomers readily aggregated into amyloid-like fibrils, while native tetramers showed little aggregation. These findings suggest that destabilization of the helically folded tetramer precedes αSyn misfolding and aggregation in Parkinson's disease and other synucleinopathies. Stabilizing the physiological tetramer could reduce αSyn pathogenicity. The study used native gel electrophoresis, Clear Native PAGE, and in vivo crosslinking to analyze αSyn in cells. αSyn was detected as a ~45-50 kDa species in various cell lines and in the frontal cortex of wild-type mice. In human red blood cells, αSyn migrated at ~55-60 kDa on Clear Native PAGE, consistent with a tetramer. Scanning transmission electron microscopy and sedimentation equilibrium analytical ultracentrifugation further supported the tetrameric structure. Circular dichroism spectroscopy showed α-helical structure in native tetramers, while recombinant monomers underwent a random coil to α-helix transition upon lipid interaction. Phospholipid analysis and mass spectrometry confirmed the absence of significant lipid association in native tetramers. The study also compared αSyn from different cell types and purification methods, finding consistent tetrameric structures. Surface plasmon resonance showed increased lipid binding by native tetramers compared to recombinant monomers. Thioflavin T fluorescence assays revealed no fibril formation by native tetramers, suggesting they are aggregation-resistant. These findings indicate that the predominant physiological form of αSyn is a helically folded tetramer, which is less prone to aggregation than monomers. The study highlights the importance of non-denaturing methods in accurately determining the native state of αSyn and suggests that stabilizing the tetramer could be a therapeutic approach for Parkinson's disease and other synucleinopathies.