Publications by year
In Press
Stephens AD, Zacharopoulou M, Moons R, Fusco G, Seetaloo N, Chiki A, Hooper PJ, Mela I, Lashuel HA, Phillips JJ, et al (In Press). Extent of N-terminus exposure by altered long-range interactions of monomeric alpha-synuclein determines its aggregation propensity.
Abstract:
Extent of N-terminus exposure by altered long-range interactions of monomeric alpha-synuclein determines its aggregation propensity
AbstractAs an intrinsically disordered protein, monomeric alpha synuclein (aSyn) constantly reconfigures and probes the conformational space. Long-range interactions across the protein maintain its solubility and mediate this dynamic flexibility, but also provide residual structure. Certain conformations lead to aggregation prone and non-aggregation prone intermediates, but identifying these within the dynamic ensemble of monomeric conformations is difficult. Herein, we used the biologically relevant calcium ion to investigate the conformation of monomeric aSyn in relation to its aggregation propensity. By using calcium to perturb the conformational ensemble, we observe differences in structure and intra-molecular dynamics between two aSyn C-terminal variants, D121A and pS129, and the aSyn familial disease mutants, A30P, E46K, H50Q, G51D, A53T and A53E, compared to wild-type (WT) aSyn. We observe that the more exposed the N-terminus and the beginning of the NAC region are, the more aggregation prone monomeric aSyn conformations become. N-terminus exposure occurs upon release of C-terminus interactions when calcium binds, but the level of exposure is specific to the aSyn mutation present. There was no correlation between single charge alterations, calcium affinity, or the number of ions bound on aSyn’s aggregation propensity, indicating that sequence or post-translation modification (PTM)-specific conformational differences between the N- and C-termini and the specific local environment mediate aggregation propensity instead. Understanding aggregation prone conformations of monomeric aSyn and the environmental conditions they form under will allow us to design new therapeutics targeted to the monomeric protein, to stabilise aSyn in non-aggregation prone conformations, by either preserving long-range interactions between the N- and C-termini or by protecting the N-terminus from exposure.
Abstract.
Seetaloo N, Kish M, Phillips JJ (In Press). HDfleX: Software for flexible high structural resolution of hydrogen/deuterium-exchange mass spectrometry data.
Abstract:
HDfleX: Software for flexible high structural resolution of hydrogen/deuterium-exchange mass spectrometry data
ABSTRACTHydrogen/deuterium-exchange mass spectrometry (HDX-MS) experiments on protein structures can be performed at three levels: (1) by enzymatically digesting labelled proteins and analyzing the peptides (bottom-up), (2) by further fragmenting peptides following digestion (middle-down), and (3) by fragmenting the intact labelled protein (top-down), using soft gas-phase fragmentation methods, such as electron transfer dissociation (ETD). However, to the best of our knowledge, the software packages currently available for the analysis of HDX-MS data do not enable the peptide- and ETD-levels to be combined – they can only be analyzed separately. Thus, we developed HDfleX – a standalone application for the analysis of flexible high structural resolution of HDX-MS data, which allows data at any level of structural resolution (intact protein, peptide, fragment) to be merged. HDfleX features rapid experimental data fitting, robust statistical significance analyses and optional methods for theoretical intrinsic calculations and a novel empirical correction for comparison between solution conditions.
Abstract.
Seetaloo N, Zacharopoulou M, Stephens AD, Kaminski Schierle GS, Phillips JJ (In Press). Local structural dynamics of alpha-synuclein correlate with aggregation in different physiological conditions.
Abstract:
Local structural dynamics of alpha-synuclein correlate with aggregation in different physiological conditions
ABSTRACTIn Parkinson’s disease and other synucleinopathies, the intrinsically disordered, presynaptic protein alpha-synuclein misfolds and aggregates. We hypothesise that the exposure of alpha-synuclein to different cellular environments, with different chemical compositions, pH and binding partners, alters its biological and pathological function by inducing changes in molecular conformation. Our custom instrumentation and software enable measurement of the amide hydrogen exchange rates of wild-type alpha-synuclein at amino acid resolution under physiological conditions, mimicking those in the extracellular, intracellular, and lysosomal compartments of cells. We characterised the aggregation kinetics and morphology of the resulting fibrils and correlate these with structural changes in the monomer. Our findings reveal that the C-terminal residues of alpha-synuclein are driving its nucleation and thus its aggregation. Furthermore, the entire NAC region and specific other residues strongly promoted elongation of fibrils. This provides new detail on our current understanding of the relationship between the local chemical environment and monomeric conformations of alpha-synuclein.
Abstract.
2023
Seetaloo N (2023). Synuclein plasticity: the Achilles’ heel of nerve function linked to the onset of Parkinson’s disease.
Abstract:
Synuclein plasticity: the Achilles’ heel of nerve function linked to the onset of Parkinson’s disease
Lewy bodies – the hallmarks of Parkinson’s disease – are majorly constituted of aggregates of the presynaptic protein alpha-synuclein. The molecular mechanism of alpha-synuclein aggregation through which it changes dramatically from a soluble disordered monomer to insoluble structured fibrils remains unknown. As an intrinsically disordered protein, alpha-synuclein does not have a specific three-dimensional structure, but rather behaves mostly as a meta-stable ensemble of highly dynamic conformers, and as such undergoes rapid kinetics, making it almost impossible to measure its conformational changes with most techniques. Millisecond amide hydrogen exchange can provide valuable insights on the dynamic behaviour of proteins, especially at flexible regions. Thus, the work in this thesis reports on the development of methods and tools for hydrogen/deuterium-exchange mass spectrometry (HDX-MS) and the application of these for the study of aSyn under physiological conditions. In the first part of this thesis, high resolution on the alpha-synuclein monomer was achieved over two dimensions: time and space. Using a novel in-house rapid- mixing quench-flow instrument, hydrogen/deuterium-exchange mass spectrometry data on alpha-synuclein on the millisecond timescale was attained. Furthermore, using a ‘soft’ gas-phase mass spectrometry fragmentation technique called Electron Transfer Dissociation, structural resolution in the protein increased. The second part of this work focuses on the development of a software, HDfleX, in an effort to primarily automate the HDX-MS workflow and allow the merging of HDX-MS data at different levels: bottom-up, middle-down and top-down. The rest of the thesis uses the tools and methods developed earlier on to explore the effects of different solution conditions (cellular compartments and salt cations) on the monomeric conformations of aSyn, and how these correlate to the different stages of aggregation and the ensuing fibril polymorphs. Altogether, the achievements in this work will allow us to better understand the plasticity of the alpha-synuclein monomer as it cycles through different local environments.
Abstract.
2022
Seetaloo N, Kish M, Phillips JJ (2022). HDfleX: Software for flexible high structural resolution of hydrogen/deuterium-exchange mass spectrometry data. Analytical Chemistry
Seetaloo N, Zacharopoulou M, Stephens AD, Kaminski Schierle GS, Phillips JJ (2022). Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry Approach to Correlate Local Structure and Aggregation in α-Synuclein. Analytical Chemistry, 94(48), 16711-16719.
Seetaloo N, Phillips JJ (2022). Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics under Physiological Conditions.
Journal of Visualized Experiments,
2022(184).
Abstract:
Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics under Physiological Conditions
Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra-and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.
Abstract.
2021
Seetaloo N, Kish M, Phillips JJ (2021). HDfleX: Software for the flexible structural resolution of hydrogen-deuterium exchange mass spectrometry data.
Abstract:
HDfleX: Software for the flexible structural resolution of hydrogen-deuterium exchange mass spectrometry data
A standalone application for the post-processing of hydrogen-deuterium exchange mass spectrometry data, including curve fitting, merging of bottom-up and middle-down data and robust statistical significance analyses, amongst others. The scientific basis and application of HDfleX has been described thoroughly in the following publication:
Seetaloo, N. Kish, M. and Phillips, J. J. (2021) ‘HDfleX: Software for flexible high structural resolution of hydrogen/deuterium-exchange mass spectrometry data’, bioRxiv, p. 2021.12.09.471740. doi: 10.1101/2021.12.09.471740.
Abstract.
2020
Stephens AD, Zacharopoulou M, Moons R, Fusco G, Seetaloo N, Chiki A, Woodhams PJ, Mela I, Lashuel HA, Phillips JJ, et al (2020). Extent of N-terminus exposure of monomeric alpha-synuclein determines its aggregation propensity.
Nature Communications,
11(1).
Abstract:
Extent of N-terminus exposure of monomeric alpha-synuclein determines its aggregation propensity
As an intrinsically disordered protein, monomeric alpha-synuclein (aSyn) occupies a large conformational space. Certain conformations lead to aggregation prone and non-aggregation prone intermediates, but identifying these within the dynamic ensemble of monomeric conformations is difficult. Herein, we used the biologically relevant calcium ion to investigate the conformation of monomeric aSyn in relation to its aggregation propensity. We observe that the more exposed the N-terminus and the beginning of the NAC region of aSyn are, the more aggregation prone monomeric aSyn conformations become. Solvent exposure of the N-terminus of aSyn occurs upon release of C-terminus interactions when calcium binds, but the level of exposure and aSyn’s aggregation propensity is sequence and post translational modification dependent. Identifying aggregation prone conformations of monomeric aSyn and the environmental conditions they form under will allow us to design new therapeutics targeted to the monomeric protein.
Abstract.