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In Parkinson's disease and other synucleinopathies, α-synuclein misfolds and aggregates. Its intrinsically disordered nature, however, causes it to adopt several meta-stable conformations stabilized by internal hydrogen bonding. Because they interconvert on short timescales, monomeric conformations of disordered proteins are difficult to characterize using common structural techniques. Few techniques can measure the conformations of monomeric α-synuclein, including millisecond hydrogen/deuterium-exchange mass spectrometry (HDX-MS). Here, we demonstrate a new approach correlating millisecond HDX-MS data with aggregation kinetics to determine the localized structural dynamics that underpin the self-assembly process in full-length wild-type monomeric α-synuclein. Our custom instrumentation and software enabled measurement of the amide hydrogen-exchange rates on the millisecond timescale for wild-type α-synuclein monomer up to residue resolution and under physiological conditions, mimicking those in the extracellular, intracellular, and lysosomal cellular compartments. We applied an empirical correction to normalize measured hydrogen-exchange rates and thus allow comparison between drastically different solution conditions. We characterized the aggregation kinetics and morphology of the resulting fibrils and correlate these with structural changes in the monomer. Applying a correlative approach to connect molecular conformation to aggregation in α-synuclein for the first time, we found that the central C-terminal residues of α-synuclein are driving its nucleation and thus its aggregation. We provide a new approach to link the local structural dynamics of intrinsically disordered proteins to functional attributes, which we evidence with new details on our current understanding of the relationship between the local chemical environment and conformational ensemble bias of monomeric α-synuclein.

The solvation shell is essential for the folding and function of proteins, but how it contributes to protein misfolding and aggregation has still to be elucidated. We show that the mobility of solvation shell H2O molecules influences the aggregation rate of the amyloid protein alpha-synuclein (alpha Syn), a protein associated with Parkinson's disease. When the mobility of H2O within the solvation shell is reduced by the presence of NaCl, alpha Syn aggregation rate increases. Conversely, in the presence CsI the mobility of the solvation shell is increased and alpha Syn aggregation is reduced. Changing the solvent from H2O to D2O leads to increased aggregation rates, indicating a solvent driven effect. We show the increased aggregation rate is not directly due to a change in the structural conformations of alpha Syn, it is also influenced by a reduction in both the H2O mobility and alpha Syn mobility. We propose that reduced mobility of alpha Syn contributes to increased aggregation by promoting intermolecular interactions.

Funder: Cambridge Commonwealth, European and International Trust, Funder: Infinitus China Ltd., The aggregation of Aβ42 is a hallmark of Alzheimer's disease. It is still not known what the biochemical changes are inside a cell which will eventually lead to Aβ42 aggregation. Thermogenesis has been associated with cellular stress, the latter of which may promote aggregation. We perform intracellular thermometry measurements using fluorescent polymeric thermometers to show that Aβ42 aggregation in live cells leads to an increase in cell-averaged temperatures. This rise in temperature is mitigated upon treatment with an aggregation inhibitor of Aβ42 and is independent of mitochondrial damage that can otherwise lead to thermogenesis. With this, we present a diagnostic assay which could be used to screen small-molecule inhibitors to amyloid proteins in physiologically relevant settings. To interpret our experimental observations and motivate the development of future models, we perform classical molecular dynamics of model Aβ peptides to examine the factors that hinder thermal dissipation. We observe that this is controlled by the presence of ions in its surrounding environment, the morphology of the amyloid peptides, and the extent of its hydrogen-bonding interactions with water. We show that aggregation and heat retention by Aβ peptides are favored under intracellular-mimicking ionic conditions, which could potentially promote thermogenesis. The latter will, in turn, trigger further nucleation events that accelerate disease progression.

Funder: Infinitus China Ltd., Conventional in vitro aggregation assays often involve tagging with extrinsic fluorophores, which can interfere with aggregation. We propose the use of intrinsic amyloid fluorescence lifetime probed using two-photon excitation and represented by model-free phasor plots as a label-free assay to characterize the amyloid structure. Intrinsic amyloid fluorescence arises from the structured packing of β-sheets in amyloids and is independent of aromatic-based fluorescence. We show that different amyloids [i.e., α-Synuclein (αS), β-Lactoglobulin (βLG), and TasA] and different polymorphic populations of αS (induced by aggregation in salt-free and salt buffers mimicking the intra-/extracellular environments) can be differentiated by their unique fluorescence lifetimes. Moreover, we observe that disaggregation of the preformed fibrils of αS and βLG leads to increased fluorescence lifetimes, distinct from those of their fibrillar counterparts. Our assay presents a medium-throughput method for rapid classification of amyloids and their polymorphs (the latter of which recent studies have shown lead to different disease pathologies) and for testing small-molecule inhibitory compounds.