Biology subjects

Dumarieh, R.

Publications and source records attributed to Dumarieh, R..

4 recordsLinked to original sources

bioRxiv2024

Structural context modulates the conformational ensemble of the intrinsically disordered amino terminus of α-synuclein

Regions of intrinsic disorder play crucial roles in biological systems, yet they often elude characterization by conventional biophysical techniques. To capture conformational distributions across different timescales, we employed a freezing approach coupled with solid-state NMR analysis. Using segmentally isotopically labeled -synuclein (-syn), we investigated the conformational preferences of the six alanines, three glycines, and a single site (L8) in the disordered amino terminus under three distinct conditions: in 8 M urea, as a frozen monomer in buffer, and within the disordered regions flanking the amyloid core. The experimental spectra varied significantly among these conditions and deviated from those of a statistical coil. In 8 M urea, monomeric -syn exhibited the most restricted conformational sampling, rarely accessing chemical shifts characteristic of -helices or {beta}-strands. In buffer, monomeric -syn showed broader conformational sampling, favoring -helical conformations and, to a lesser extent, random coil states. Notably, amino acids in the disordered regions flanking the amyloid core demonstrated the most extensive conformational sampling, with broad peaks encompassing the entire range of possible chemical shifts and a marked preference for highly extended {beta}-strand conformations. Collectively, this work demonstrates that intrinsically disordered regions exhibit distinct conformational preferences, which are influenced not only by the chemical environment but also by the conformations of adjacent protein sequences. The differences in the conformational ensembles of the disordered amino terminus may explain why the monomer and the amyloid form of -syn interact with different biomolecules inside cells.

biophysics↗

bioRxiv2024

In cell NMR reveals cells selectively amplify and structurally remodel amyloid fibrils

Amyloid forms of -synuclein adopt different conformations depending on environmental conditions. Although advances in structural biology have accelerated fibril characterization, it remains unclear which conformations predominate in biological settings because current approaches typically require fibrils to be isolated from their native environments. In addition, these approaches provide limited information about flanking flexible regions. Here, using a defined polymorphic seed population and quantitative in-cell NMR, we show that propagation in intact cellular environments--but not in vitro buffer or crowded cellular lysates--reshapes the conformational ensemble of -synuclein fibrils. In vitro and in cellular lysates, both amyloid-core structure and the conformational preferences of flanking intrinsically disordered regions of the seed are faithfully propagated. In contrast, propagation inside intact cells selectively amplifies the amyloid-core conformer that is minor in vitro, increases its molecular order, and remodels conformational preferences in the flanking disordered region. These results demonstrate that cellular organization plays a decisive role in determining amyloid structure and that biologically relevant conformations cannot be inferred from purified or lysate systems alone.

biophysics↗

bioRxiv2023

Spatially resolved DNP-assisted NMR illuminates the conformational ensemble of α-synuclein in intact viable cells.

The protein -syn adopts a wide variety of conformations including an intrinsically disordered monomeric form and an -helical rich membrane-associated form that is thought to play an important role in cellular membrane processes. However, despite the high affinity of -syn for membranes, evidence that the -helical form is adopted inside cells has been indirect. DNP-assisted solid state NMR on frozen cellular samples can report on protein conformations inside cells. Moreover, by controlling the distribution of the DNP polarization agent throughout the cellular biomass, such experiments can provide quantitative information upon the entire structural ensemble or provide information about spatially resolved sub-populations. Using DNP-assisted magic angle spinning (MAS) NMR we establish that purified -syn in the membrane-associated and intrinsically disordered forms have distinguishable spectra. We then introduced isotopically labeled monomeric -syn into cells. When the DNP polarization agent is dispersed homogenously throughout the cell, we found that a minority of the -syn inside cells adopted a highly -helical rich conformation. When the DNP polarization agent is peripherally localized, we found that the -helical rich conformation predominates. Thus, we provide direct evidence that -helix rich conformations of -syn are adopted near the cellular periphery inside cells under physiological conditions. Moreover, we demonstrate how selectively altering the spatial distribution of the DNP polarization agent can be a powerful tool to observe spatially distinct structural ensembles. This approach paves the way for more nuanced investigations into the conformations that proteins adopt in different areas of the cell.

biophysics↗

bioRxiv2022

The conformational ensemble of an intrinsically disordered protein explains peak shapes under DNP conditions

Elucidating the conformational preferences of regions of intrinsic disorder in biologically relevant contexts represents a frontier of structural biology. The sensitivity enhancements conferred by DNP enable structural studies of proteins in native contexts by MAS NMR. However, DNP requires low temperatures which results in broad peaks, particularly for for regions of intrinsic disorder. We describe an approach to predict and interpret peak shapes for frozen regions of intrinsic disorder in terms of dihedral angle populations. We demonstrate the method using the protein a-synuclein. This approach can be used to obtain experimental structural restraints for regions of intrinsic disorder in both simplified and biological settings, providing information that eludes characterization by diffraction-based methods as well as solution-state NMR spectroscopy and molecular dynamics due to molecular size limitations.

biophysics↗