Characterization of Semisynthetic and Naturally Nα-Acetylated α-Synuclein in Vitro and in Intact Cells

Abstract

Background: How N-terminal acetylation affects the structure and function of ␣-syn remains unknown. Results: N-terminally acetylated and WT ␣-syn are unfolded monomers and exhibit similar aggregation and cellular properties. Conclusion: ␣-syn N-terminal acetylation does not dramatically affect its structure or oligomerization state in vitro and in intact cells. Significance: Recombinant nonacetylated or N ␣ -acetylated ␣-syn remains suitable for ␣-syn biophysical studies.

N-terminal Acetylation Stabilizes N-terminal Helicity in Lipid- and Micelle-bound α-Synuclein and Increases Its Affinity for Physiological Membranes

“…␣-Carbon secondary shifts for Ac-aSyn in the presence of 100 mM BOG (Fig. 4C) show significant positive deviations for the first 30 residues, whereas the remainder of the protein exhibits secondary shifts very similar to those previously determined for free Ac-aSyn (30). The amplitude of the N-terminal secondary shifts decreases from just above 2 ppm to just below 0.5 ppm.…”

“…Protein Expression and Purification-N-terminally acetylated and unmodified aSyn were produced as previously reported (30,35). Protein uniformly labeled with 15 N and 13 C, 15 N for heteronuclear NMR experiments was produced by the media swap method (37).…”

“…Concentrations of BOG and lipid vesicles were determined using the intensity of alkyl chain peaks in onedimensional proton NMR spectra. R 2 relaxation rates were measured through a series of 9 HSQC-based T 2 experiments with relaxation time delays of 10,10,10,30,30,50,70,90, and 110 ms. The intensity decay of each cross-peak was fit to a single exponential decay function using internal NMRViewJ fitting module.…”

“…Nevertheless, N-terminal acetylation does influence the secondary structure of the protein (30,32,33). Because synuclein undergoes a disorder-to-helix transition upon binding to membranes and membrane mimetics (34,35), it might be expected that the effects of N-terminal acetylation on helical secondary structure could alter aSyn-membrane interactions.…”

Remodeling of Lipid Vesicles into Cylindrical Micelles by α-Synuclein in an Extended α-Helical Conformation

“…It has long been known that ␣S is an intrinsically disordered protein in solution (28). This view has recently been challenged by suggesting that the protein exists as a natively folded tetramer (30,31), but further experimentation, including in-cell NMR (29,72), indicate that ␣S is an unfolded monomer in vivo. Upon long incubation, it passes through toxic oligomeric states to reach an amyloid conformation (32), rich in parallel in-register cross-␤ structure (33,34), which is considered to be its state in Lewy body accumulations.…”