Room-temperature in-cell EPR spectroscopy: alpha-Synuclein disease variants remain intrinsically disordered in the cell

Human alpha-Synuclein (aS), implicated in Parkinson's disease, adopts a rich variety of different conformations depending on the macromolecular context. In order to unravel its pathophysiological role, monitoring its intracellular conformational state and identifying differences for the disease variants is crucial. Here, we present an intracellular spectroscopy approach based on a systematic spin-labeling site-scan in combination with intracellular electron paramagnetic resonance spectroscopy determining conformations on a molecular scale. A quantitative and model-based data analysis revealed that the vast majority of aS, be it wild-type or disease variants A30P or A53T, exists in the monomeric intrinsically disordered form in the cell.