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

Abstract

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.

Determining the Location of the α-Synuclein Dimer Interface Using Native Top-Down Fragmentation and Isotope Depletion-Mass Spectrometry

“…These findings suggest that αSyn can form intermolecular interactions between the C-termini of two monomer units, within a dimer species. In addition, a recent hydrogen–deuterium exchange MS investigation observed that peptides primarily within the C-terminus and the NAC domain of the protein are important in the formation of higher order species of αSyn . Interestingly, none of the identified familial mutations associated with PD occur within this region of the protein.…”

Online Fully Automated System for Hydrogen/Deuterium-Exchange Mass Spectrometry with Millisecond Time Resolution

“…The new “ms2min” system developed here has significant merits from a technical standpoint: considerable potential for HDX-MS resolved at the millisecond level has been demonstrated previously, for example, applied to the study of protein folding. 2 The ms2min system stands to be particularly impactful in the future study of intrinsically disordered proteins/regions 53 , 55 and for emerging time-resolved (i.e., non-equilibrium) protein conformational dynamics, for example, applied to transmission electron microscopy lactamase catalysis, 5 given its software control over single millisecond differences in the labeling time and online full automation. Given a recently proposed framework for temperature correction to quantitatively expand the time window of HDX-MS measurements, the ms2min instrument should be capable of sub-millisecond timepoints at a reference temperature of 298 K. 56 The recent development of sub-zero LC for bottom-up HDX-MS will expand the technique to vastly more complicated samples, such as blood plasma, crude cell lysate, and cell culture media.…”

Ion Mobility Mass Spectrometry Unveils Global Protein Conformations in Response to Conditions that Promote and Reverse Liquid–Liquid Phase Separation

“…26 In addition to nMS, hydrogen/deuterium exchange MS can also reveal the effect of salts, metal ions, and buffers on the metastable conformations of IDPs. 27,28 The hybrid method of IM−MS adds an extra dimension of analysis by separating protein ions on the basis of their overall size, meaning that multiple conformations can be separated from one m/z ratio. Larger conformations travel slower through an IM drift region, due to an increased number of collisions with gas molecules.…”

“…Toward analyzing proteins from more physiological conditions, Sharon and co-workers have shown considerable success with nMS of overexpressed proteins in crude cell lysates . In addition to nMS, hydrogen/deuterium exchange MS can also reveal the effect of salts, metal ions, and buffers on the metastable conformations of IDPs. , …”