The structural heterogeneity of α-synuclein is governed by several distinct subpopulations with interconversion times slower than milliseconds

Abstract

The intrinsically disordered protein, α-synuclein, implicated in synaptic vesicle homeostasis and neurotransmitter release, is also associated with several neurodegenerative diseases. The different roles of α-synuclein are characterized by distinct structural states (membrane-bound, dimer, tetramer, oligomer, and fibril), which are originated from its various monomeric conformations. The pathological states, determined by the ensemble of α-synuclein monomer conformations and dynamic pathways of interconversion between dominant states, remain elusive due to their transient nature. Here, we use inter-dye distance distributions from bulk time-resolved Förster resonance energy transfer as restraints in discrete molecular dynamics simulations to map the conformational space of the α-synuclein monomer. We further confirm the generated conformational ensemble in orthogonal experiments utilizing far-UV circular dichroism and cross-linking mass spectrometry. Single-molecule protein-induced fluorescence enhancement measurements show that within this conformational ensemble, some of the conformations of α-synuclein are surprisingly stable, exhibiting conformational transitions slower than milliseconds. Our comprehensive analysis of the conformational ensemble reveals essential structural properties and potential conformations that promote its various functions in membrane interaction or oligomer and fibril formation.

Structural and Dynamic Insights Into α-Synuclein Dimer Conformations

“…We hypothesize that like in the case of the free-form monomer 30,31 the αSyn dimer may exhibit distinct subpopulations with different characteristic fluorescence lifetimes. To gain insights on the dimer formation we performed smPIFE measurements.…”

“…To investigate the structural changes associated with the formation of an αSyn dimer, we perform sulfo-Cy3 (sCy3)-based intra-molecular time-resolved smPIFE measurements 30,31 . The uniqueness of this dye is that it exhibits excited-state isomerization between a bright trans isomer and a dark cis isomer, leading to an overall low fluorescence quantum yield.…”

“…CL-MS has emerged as a robust method for studying protein structures and assemblies 38–41 . Recently, CL-MS has been used to study small disordered proteins, such as the free-form αSyn monomer and assisted in elucidating its ensemble structure 31 . One of the limitations of CL-MS is the inability to distinguish between inter- and intra-molecular cross-links in homodimers.…”

“…Here, we demonstrate αSyn exhibits predominantly a monomer-dimer equilibrium in the nanomolars (nM) and up to a few micromolars (μM), which is below the αSyn concentrations in which αSyn exhibits aggregation that leads to fibril formation 13 . Using single-molecule photo-isomerization related fluorescence enhancement (smPIFE; commonly known as protein-induced fluorescence enhancement) 28–31 , we demonstrate dimer formation is associated with structural changes that lead to specific changes in amino acid steric hindrances. Nevertheless, at a high dimer fraction, the smPIFE results still exhibit several different conformations should exist, with transitions between them as slow as milliseconds.…”

Conformational Ensemble of Monomeric α-Synuclein in Aqueous and Crowded Environments as revealed by Markov State Model

“…They revealed the presence of long-range tertiary interactions between the negatively charged C-terminal tail and the hydrophobic NAC or N-terminal residues; these conformations are potentially autoinhibitory towards oligomerization and aggregation. Moreover, single molecule experiments 45,46 and molecular simulations [47][48][49] have also investigated the conformational dynamics of monomeric αS and measured the timescales of secondary and tertiary structure formation relevant to the pathological αS fibrils.…”