Biology subjects

Kragelj, J.

Publications and source records attributed to Kragelj, J..

6 recordsLinked to original sources

bioRxiv2025

Fast MAS NMR Spectroscopy Can Identify G-Quartets and Double-Stranded Structures in Aggregates Formed by GGGGCC RNA Repeats

The expansion of GGGGCC repeats within the C9orf72 gene has been linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In neurons from patients with expanded repeats, C9orf72 GGGGCC repeat RNA predominantly forms nuclear foci, and in vitro, repeat-containing RNA can self-aggregate. Two structural motifs have been proposed to provide the interstrand interactions that drive aggregation: G-quartet (G4) structures and double-strand interactions with GG mismatches. Using in vitro transcribed RNA with pathologically relevant number of repeats, we were able to form gel-like aggregates suitable for investingation using fast MAS NMR spectroscopy. This approach enabled us to characterize the dominant interstrand interactions within the RNA gels. Both Watson-Crick and Hoogsteen base pairs were identified in RNA gels formed by RNA with 48 GGGGCC repeats. Their relative abundance shifted upon reconstitution in the presence of different divalent cations or nuclear extracts, underscoring the dynamic equilibrium between G-quadruplex and duplex interactions in GGGGCC RNA aggregation.

biophysics↗

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↗

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↗

bioRxiv2023

DNP-assisted solid-state NMR enables detection of proteins at nanomolar concentrations in fully protonated cellular environments

With the sensitivity enhancements conferred by dynamic nuclear polarization (DNP), magic angle spinning (MAS) solid state NMR spectroscopy experiments can attain the necessary sensitivity to detect very low concentrations of proteins. This potentially enables structural investigations of proteins at their endogenous levels in their biological contexts where their native stoichiometries with potential interactors is maintained. Yet, even with DNP, experiments are still sensitivity limited. Moreover, when an isotopically-enriched target protein is present at physiological levels, which typically range from low micromolar to nanomolar concentrations, the isotope content from the natural abundance isotopes in the cellular milieu can outnumber the isotope content of the target protein. Using isotopically enriched yeast prion protein, Sup35NM, diluted into natural abundance yeast lysates, we optimized sample composition we find that modest cryoprotectant concentrations and fully protonated environments support efficient DNP. We experimentally validated theoretical calculations of the limit of specificity for an isotopically enriched protein in natural abundance cellular milieu. We establish that, using pulse sequences that are selective for adjacent NMR-active nuclei, proteins can be specifically detected in cellular milieu at concentrations in the hundreds of nanomolar. Finally, we find that maintaining native stoichiometries of the protein of interest to the components of the cellular environment may be important for proteins that make specific interactions with cellular constituents.

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↗

bioRxiv2021

In-Cell Sensitivity-Enhanced NMR of Intact Living Mammalian Cells

NMR has the resolution and specificity to determine atomic-level protein structures of isotopically-labeled proteins in complex environments and, with the sensitivity gains conferred by dynamic nuclear polarization (DNP), NMR has the sensitivity to detect proteins at their endogenous concentrations. However, DNP sensitivity enhancements are critically dependent on experimental conditions and sample composition. While some of these conditions are theoretically compatible with cellular viability, the effects of others on cellular sample integrity are unknown. Uncertainty about the integrity of cellular samples limits the utility of experimental outputs. Using several measures, we establish conditions that support DNP enhancements that can enable detection of micromolar concentrations of proteins in experimentally tractable times that are compatible with cellular viability. Taken together, we establish DNP assisted MAS NMR as a technique for structural investigations of biomolecules in intact viable cells that can be phenotyped both before and after NMR experiments. ClassificationBiophysics and Structural Biology

biophysics↗