Biology subjects

Seetaloo, N.

Publications and source records attributed to Seetaloo, N..

5 recordsLinked to original sources

bioRxiv2024

Local ionic conditions modulate the aggregation propensity and influence the structural polymorphism of alpha-synuclein

Parkinsons Disease (PD) is characterized by the aggregation of alpha-synuclein (aSyn), a presynaptic protein that transitions from a disordered monomer into beta-sheet rich amyloid fibrils. The precise triggers and mechanisms underlying aSyn misfolding and aggregation remain unclear, hindering the development of effective therapeutics. Monomeric aSyn is an intrinsically disordered protein (IDP) with high conformational flexibility. Local environmental factors, such as ion concentrations, can influence the conformational ensemble of aSyn, impacting its aggregation propensity and resulting in fibril polymorphism. In this study, we explore the impact of physiologically relevant ions, mainly Ca2+ and Na+, on the aggregation kinetics, monomer structural dynamics, and fibril polymorphism of aSyn. Using ThT fluorescence assays, we demonstrate that all ions accelerate aSyn aggregation, with Ca2+ having the most significant effect. Using Heteronuclear Single Quantum Correlation Nuclear Magnetic Resonance (1H-15NHSQC NMR) spectroscopy, we validate the specific binding of Ca2+ ions at the C-terminus, whereas Na+ ions display non-specific interactions along the sequence of aSyn. Small-angle neutron scattering (SANS) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) further reveal that Na+ and Ca2+ induce distinct conformational changes in the aSyn monomer, with Na+ leading to more extended structures and Ca2+ promoting a moderate extension of the protein. Molecular dynamics simulations (MD) corroborate these findings, showing that Na+ ions increase the proteins extension, particularly between the non-amyloid beta component (NAC) region and the C-terminus, whereas Ca2+ ions bias the ensemble towards a more moderately elongated structure. Using MD, we further investigate the local environment and in particular the solvent effect and show the water persistence times in the hydration shell are also increased in the presence of Ca2+ ions, indicating that the aggregation propensity of the monomer is due to a combination of conformational bias of the monomer and solvent mobility. Atomic force microscopy (AFM) of aSyn fibrils formed under these different ionic conditions reveal distinct fibril polymorphs, suggesting that ion-induced conformational biases in the monomer contribute to the diversity of fibril structures. Collectively, these findings underscore the pivotal influence of the local ionic milieu in shaping the structure and aggregation propensity of aSyn, thus offering valuable insights into the molecular underpinnings of PD and potential therapeutic avenues aimed at manipulating aSyn conformational dynamics.

biophysics↗

bioRxiv2023

DNA binding and bridging by human CtIP in the healthy and diseased states

The human DNA repair factor CtIP helps to initiate the resection of double-stranded DNA breaks for repair by homologous recombination, in part through its ability to bind and bridge DNA molecules. However, CtIP is a natively disordered protein that bears no apparent similarity to other DNA-binding proteins and so the structural basis for these activities remains unclear. In this work, we have used bulk DNA binding, single molecule tracking, and DNA bridging assays to study wild-type and variant CtIP proteins to better define the DNA binding domains and the effects of mutations associated with inherited human disease. Our work identifies a monomeric DNA-binding domain in the C-terminal region of CtIP. CtIP binds non-specifically to DNA and can diffuse over thousands of nucleotides. CtIP-mediated bridging of distant DNA segments is observed in single-molecule magnetic tweezers experiments. However, we show that binding alone is insufficient for DNA bridging, which also requires tetramerization via the N-terminal domain. Variant CtIP proteins associated with Seckel and Jawad syndromes display impaired DNA binding and bridging activities. The significance of these findings in the context of facilitating DNA break repair is discussed. Significance StatementCtIP helps to repair broken chromosomes through its ability to bind and bridge DNA molecules. We studied the structural and biochemical basis for these activities and how they are affected by hereditary CtIP mutations associated with developmental disorders. We discovered a minimal domain in the C-terminal region of CtIP which supports DNA binding as a monomer. DNA binding is non-specific and facilitates 1D diffusion, but binding alone is insufficient for intermolecular tethering of DNA molecules which requires tetramerization of CtIP via N-terminal coiled-coil domains. All disease variants tested displayed impaired DNA bridging activity. These results have important implications for understanding the role of CtIP as a hub protein for DNA break repair and its dysfunction in human disease.

biochemistry↗

bioRxiv2022

Local structural dynamics of alpha-synuclein correlate with aggregation in different physiological conditions

In Parkinsons 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.

molecular biology↗

bioRxiv2021

HDfleX: Software for flexible high structural resolution of hydrogen/deuterium-exchange mass spectrometry data

Hydrogen/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.

biophysics↗

bioRxiv2019

Extent of N-terminus exposure by altered long-range interactions of monomeric alpha-synuclein determines its aggregation propensity

As 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 aSyns 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.

biophysics↗