[NMR paper] NMR Observation of ?-Synuclein Membrane Interaction by Monitoring Acetylation Reactivity of its Lysine Sidechains.

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NMR Observation of ?-Synuclein Membrane Interaction by Monitoring Acetylation Reactivity of its Lysine Sidechains.

Related Articles NMR Observation of ?-Synuclein Membrane Interaction by Monitoring Acetylation Reactivity of its Lysine Sidechains.

Biochemistry. 2016 Jul 25;

Authors: Lee JH, Ying J, Bax A

Abstract
The interaction between ?-synuclein (?S) protein and lipid membranes is key to its role in synaptic vesicle homeostasis, and plays a role in initiating fibril formation which is implicated in Parkinson's disease. The natural state of ?S inside the cell is generally believed to be intrinsically disordered, but chemical cross-linking experiments provided evidence for a tetrameric arrangement, which was reported to be rich in ?-helical secondary structure based on circular dichroism (CD). Cross-linking relies on chemical modification of the protein's Lys C? amino groups, commonly by glutaraldehyde, or by disuccinimidyl glutarate (DSG), with the latter agent preferred for cellular assays. We used ultra-high resolution homonuclear decoupled NMR experiments to probe the reactivity of the 15 ?S Lys residues towards N-succinimidyl-acetate, effectively half the DSG cross linker, which results in acetylation of Lys. The intensities of both sidechain and backbone amide signals of acetylated Lys residues provide direct information on the reactivity, showing a difference by a factor 2.5 between the most reactive (K6) and the least reactive residue (K102). The presence of phospholipid vesicles decreases reactivity of most Lys residues by up to an order of magnitude at high lipid:protein stoichiometries (500:1), but only weakly at low ratios. The decrease of Lys reactivity is found to be impacted by lipid composition, even for vesicles that yield similar ?S CD signatures. Our data provide new insight on the ?S-bilayer interaction, including the pivotal state where the available lipid surface is limited. Protection of Lys C? amino groups by ?S-bilayer interaction will strongly impact quantitative interpretation of DSG cross-linking experiments.


PMID: 27455358 [PubMed - as supplied by publisher]



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