Spatially resolved DNP-assisted NMR illuminates the conformational ensemble of α-synuclein in intact viable cells

The protein α-syn adopts a wide variety of conformations including an intrinsically disordered monomeric form and an α-helical rich membrane-associated form that is thought to play an important role in cellular membrane processes. However, despite the high affinity of α-syn for membranes, evidence that the α-helical form of α-syn is adopted inside cells has thus far been indirect. In cell DNP-assisted solid state NMR on frozen samples has the potential to report directly on the entire conformational ensemble. Moreover, because the DNP polarization agent can be dispersed both homogenously and inhomogenously throughout the cellular biomass, in cell DNP-assisted solid state NMR experiments can report either quantitatively upon the structural ensemble or can preferentially report upon the structural ensemble with a spatial bias. Using DNP-assisted MAS NMR we establish that the spectra of purified α-syn in the membrane-associated and intrinsically disordered forms have distinguishable spectra. When the polarization agent is introduced into cells by electroporation and dispersed homogenously, a minority of the α-syn inside HEK293 cells adopts a highly α-helical rich conformation. Alteration of the spatial distribution of the polarization agent preferentially enhances the signal from molecules nearer to the cellular periphery, thus the α-helical rich population is preferentially adopted toward the cellular periphery. This demonstrates how selectively altering the spatial distribution of the DNP polarization agent can be a powerful tool for preferential reporting on specific structural ensembles, paving the way for more nuanced investigations into the conformations that proteins adopt in different areas of the cell.