Conformational Dynamics of α-Synuclein during the Interaction with Phospholipid Nanodiscs by Millisecond Hydrogen–Deuterium Exchange Mass Spectrometry

Abstract

Both normal and pathological functions of αsynuclein (αSN), an abundant protein in the central and peripheral nervous system, have been linked to its interaction with membrane lipid bilayers. The ability to characterize structural transitions of αSN upon membrane complexation will clarify molecular mechanisms associated with αSN-linked pathologies, including Parkinson's disease (PD), multiple systems atrophy, and other synucleinopathies. In this work, time-resolved electrospray ionization hydrogen/deuterium exchange mass spectrometry (TRESI-HDX-MS) was employed to acquire a detailed picture of αSN's conformational transitions as it undergoes complexation with nanodisc membrane mimics with different headgroup charges (zwitterionic DMPC and negative POPG). Using this approach, αSN interactions with DMPC nanodiscs were shown to be rapid exchanging and to have little impact on the αSN conformational ensemble. Interactions with nanodiscs containing lipids known to promote amyloidogenesis (e.g., POPG), on the other hand, were observed to induce substantial and specific changes in the αSN conformational ensemble. Ultimately, we identify a region corresponding residues 19−28 and 45−57 of the αSN sequence that is uniquely impacted by interactions with "amyloidogenic" lipid membranes, supporting the existing "broken-helix" model for αsynuclein/membrane interactions, but do not detect a "helical extension" that is also thought to play a role in αSN aggregation.

Subsecond Time-Resolved Mass Spectrometry in Dynamic Structural Biology

“…The study observed that global refolding corresponding to the formation of secondary structural elements occurred relatively late in the pathway at 10 s. 200 Finally, work from Oganesyan, Wilson, and co-workers explored the changes in conformational dynamics associated with the interaction between alpha-synuclein (a Parkinson's-linked protein) and lipid nanodiscs with different compositions. 201 The results were mostly confirmatory of previous NMR-based studies on this system but were nonetheless the first application of timeresolved HDX-MS to characterize protein lipid nanodisc interactions, and they provided critical orthogonal support for proposed mechanisms of pathogenic alpha-synuclein aggregation in this challenging system.…”

Lipids and EGCG Affect α-Synuclein Association and Disruption of Nanodiscs

“…These data indicate that free α-syn can bind zwitterionic lipids to a small degree, but α-syn does not directly associate with the zwitterionic membrane surface. Prior research has also observed minimal interactions with zwitterionic lipids. , …”

“…Prior research has also observed minimal interactions with zwitterionic lipids. 45,46 In contrast, α-syn directly associated with anionic DMPG nanodiscs. Macromolecular mass defect analysis revealed the stoichiometry of associations of α-syn with DMPG nanodiscs (Figure 1D,E) as 0:1, 1:1, and 2:1 α-syn:nanodisc at lower concentrations.…”

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry Approach to Correlate Local Structure and Aggregation in α-Synuclein

“…Due to its rapidly interconverting conformations, the structural dynamics of monomeric aSyn is intractable by most structural biology techniques. Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) is one of the few techniques capable of capturing such conformational information on aSyn at high structural resolution. , Previous studies on aSyn using HDX-MS have shown that it exchanges on the millisecond timescale and as such requires specialized instrumentation. , Here, we obtained data at high structural and temporal resolution for the aSyn monomer under physiologically relevant solution conditions. This was achieved by HDX-MS on the millisecond timescale coupled with a gas-phase “soft fragmentation” technique, electron-transfer dissociation (ETD).…”

“… 16 , 17 Previous studies on aSyn using HDX-MS have shown that it exchanges on the millisecond timescale and as such requires specialized instrumentation. 18 , 19 Here, we obtained data at high structural and temporal resolution for the aSyn monomer under physiologically relevant solution conditions. This was achieved by HDX-MS on the millisecond timescale coupled with a gas-phase “soft fragmentation” technique, electron-transfer dissociation (ETD).…”