In dopaminergic neurons, a-synuclein (aS) partitions between a disordered cytosolic state and a lipid-bound state. Binding of aS to membrane phospholipids is implicated in its functional role of synaptic regulation, but also impacts fibril formation associated with Parkinson's disease. A 2011 study by Selkoe et al reported that if aS is expressed in mammalian cells and purified without a heat denaturation step, it adopts a stable tetrameric helical structure. We developed this expression system but were unable to duplicate their findings. However, we found by high-resolution NMR spectroscopy and circular dichroism (CD) measurements, that the N-terminal acetylation which occurs in mammalian cells impacts the protein's structure and dynamics in free solution and also affects the protein's membrane binding properties. While no tetrameric form of acetylated aS could be isolated, N-terminal acetylation resulted in chemical shift perturbations of the first 12 residues of the protein which progressively decreased with distance from the N-terminus. The directions of the chemical shift changes and small changes in backbone 3JHH couplings are consistent with an increase in alpha-helicity of the first six residues of aS, although a high degree of dynamic conformational disorder remains and the helical structure is sampled less than 20%. Chemical shift and 3JHH data for the intact protein are virtually indistinguishable from those recorded for the corresponding N-terminally acetylated and non-acetylated 15-residue synthetic peptides. An increase in alpha-helicity at the N-terminus of aS is supported by CD data on the acetylated peptide, and by weak medium-range NOE contacts indicative of alpha-helical character. The remainder of the protein has chemical shift values that are very close to random coil values and indistinguishable between the two forms of the protein. No significant difference in the fibrillation kinetics were observed between acetylated and non-acetylated aS. However, the lipid binding properties of aS are strongly impacted by acetylation, and exhibit distinct behavior for the first 12 residues, indicative of an initiation role for the N-terminal residues in an """"""""initiation-elongation"""""""" process of binding to the membrane. A novel method for probing the membrane binding of alpha-synuclein has been developed which relies on the spontaneous oxidation of its Met residues when a small fraction of the lipids in small unilamellar vesicles (SUVs) contain peroxidized alkane chains. Probing of the rates of oxidation of different Met residues in synuclein revealed a strong degree of cooperativity in the binding of the N-terminal 50 residues of the protein to the membrane surface.

Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2013
Total Cost
$676,793
Indirect Cost
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Alderson, T Reid; Bax, Ad (2016) Parkinson's disease: Disorder in the court. Nature 530:38-9
Lee, Jung Ho; Ying, Jinfa; Bax, Ad (2016) Nuclear Magnetic Resonance Observation of α-Synuclein Membrane Interaction by Monitoring the Acetylation Reactivity of Its Lysine Side Chains. Biochemistry 55:4949-59
Roche, Julien; Ying, Jinfa; Shen, Yang et al. (2016) ARTSY-J: Convenient and precise measurement of (3)JHNHα couplings in medium-size proteins from TROSY-HSQC spectra. J Magn Reson 268:73-81
Lee, Jung Ho; Ying, Jinfa; Bax, Ad (2016) Quantitative evaluation of positive Ï• angle propensity in flexible regions of proteins from three-bond J couplings. Phys Chem Chem Phys 18:5759-70
Werner-Allen, Jon W; DuMond, Jenna F; Levine, Rodney L et al. (2016) Toxic Dopamine Metabolite DOPAL Forms an Unexpected Dicatechol Pyrrole Adduct with Lysines of α-Synuclein. Angew Chem Int Ed Engl 55:7374-8
Roche, Julien; Ying, Jinfa; Bax, Ad (2016) Accurate measurement of (3)J(HNHα) couplings in small or disordered proteins from WATERGATE-optimized TROSY spectra. J Biomol NMR 64:1-7
Lee, Jung Ho; Li, Fang; Grishaev, Alexander et al. (2015) Quantitative residue-specific protein backbone torsion angle dynamics from concerted measurement of 3J couplings. J Am Chem Soc 137:1432-5
Mantsyzov, Alexey B; Shen, Yang; Lee, Jung Ho et al. (2015) MERA: a webserver for evaluating backbone torsion angle distributions in dynamic and disordered proteins from NMR data. J Biomol NMR 63:85-95
Mantsyzov, Alexey B; Maltsev, Alexander S; Ying, Jinfa et al. (2014) A maximum entropy approach to the study of residue-specific backbone angle distributions in α-synuclein, an intrinsically disordered protein. Protein Sci 23:1275-90
Maltsev, Alexander S; Chen, Jue; Levine, Rodney L et al. (2013) Site-specific interaction between ýý-synuclein and membranes probed by NMR-observed methionine oxidation rates. J Am Chem Soc 135:2943-6

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