using-solution-nmr-to-characterize-biomolecular-condensates-under

Using Solution NMR to Characterize Biomolecular Condensates Under Biphasic Conditions

Biomolecular condensates formed through liquid&#8211;liquid phase separation (LLPS) organize the intracellular environment and regulate diverse ...

stabilizing-protein-proteinrna-condensates-low-melting-agarose

Stabilizing Protein and Protein&#8212;RNA Condensates in Low-Melting Agarose Hydrogels for Quantitative Biophysical Measurements

Biomolecular condensates formed by liquid&#8211;liquid phase separation are increasingly recognized as fundamental organizational units of the cell, with ...

ETH Zurich

ETH Zürich

Phase separated macromolecules play essential roles in many biological and synthetic systems. Physical characterization of these systems can be challenging because of limited sample volumes, particularly for phase-separated proteins. Here, we demonstrate that a classic method for measuring the surface tension of liquid droplets, based on the analysis of the shape of a sessile droplet, can be effectively scaled down to measure the interfacial tension between a macromolecule-rich droplet phase and its co-existing macromolecule-poor continuous phase. The connection between droplet shape and surface tension relies on the density difference between the droplet and its surroundings. This can be determined with small sample volumes in the same setup by measuring the droplet sedimentation velocity. An interactive MATLAB script for extracting the capillary length from a droplet image is included in the ESI.

Surface tensiometry of phase separated protein and polymer droplets by the sessile drop method.

Many RNA-binding proteins undergo liquid-liquid phase separation, which underlies the formation of membraneless organelles, such as stress granules and P-bodies. Studies of the molecular mechanism of phase separation in vitro are hampered by the coalescence and sedimentation of organelle-sized droplets interacting with glass surfaces. Here, we demonstrate that liquid droplets of fused in sarcoma (FUS)-a protein found in cytoplasmic aggregates of amyotrophic lateral sclerosis and frontotemporal dementia patients-can be stabilized in vitro using an agarose hydrogel that acts as a cytoskeleton mimic. This allows their spectroscopic characterization by liquid-phase NMR and electron paramagnetic resonance spectroscopy. Protein signals from both dispersed and condensed phases can be observed simultaneously, and their respective proportions can be quantified precisely. Furthermore, the agarose hydrogel acts as a cryoprotectant during shock-freezing, which facilitates pulsed electron paramagnetic resonance measurements at cryogenic temperatures. Surprisingly, double electron-electron resonance measurements revealed a compaction of FUS in the condensed phase.

NMR and EPR reveal a compaction of the RNA-binding protein FUS upon droplet formation.

Function of intrinsically disordered proteins may depend on deviation of their conformational ensemble from that of a random coil. Such deviation may be hard to characterize and quantify, if it is weak. We explored the potential of distance distributions between spin labels, as they can be measured by electron paramagnetic resonance techniques, for aiding such characterization. On the example of the intrinsically disordered N-terminal domain 1-267 of fused in sarcoma (FUS) we examined what such distance distributions can and cannot reveal on the random-coil reference state. On the example of the glycine-rich domain 188-320 of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) we studied whether deviation from a random-coil ensemble can be robustly detected with 19 distance distribution restraints. We discuss limitations imposed by ill-posedness of the conversion of primary data to distance distributions and propose overlap of distance distributions as a fit criterion that can tackle this problem. For testing consistency and size sufficiency of the restraint set, we propose jack-knife resampling. At current desktop computers, our approach is expected to be viable for domains up to 150 residues and for between 10 and 50 distance distribution restraints.

Characterization of Weak Protein Domain Structure by Spin-Label Distance Distributions.

Liquid-liquid phase separation of RNA-binding proteins underlies the formation of membraneless organelles, whose composition is dynamic and whose existence may be transient. These organelles are involved in regulation of RNA processing and translation and, if they behave abnormally, in pathologies. Because disorder phenomena are essential in their formation and dynamics, established methodology is insufficient for characterizing their structure. In this review, we consider the current and potential contribution of NMR and EPR spectroscopy to the understanding of structure and dynamics of phase-separating RNA-binding proteins in, both, their dispersed and condensed state in vitro. We discuss which experiments are applicable under what conditions and which information can be obtained from them. Because for these phenomena, the accessible information depends crucially on metastable phase equilibria, we also consider aspects of sample preparation for NMR and EPR experiments.

Structural biology of RNA-binding proteins in the context of phase separation: What NMR and EPR can bring?

Many membraneless organelles are liquid-like domains that form inside the active, viscoelastic environment of living cells through phase separation. To investigate the potential coupling of phase separation with the cytoskeleton, we quantify the structural correlations of membraneless organelles (stress granules) and cytoskeletal filaments (microtubules) in a human-derived epithelial cell line. We find that microtubule networks are substantially denser in the vicinity of stress granules. When microtubules are depolymerized, the sub-units localize near the surface of the stress granules. We interpret these data using a thermodynamic model of partitioning of particles to the surface and bulk of the droplets. In this framework, our data are consistent with a weak (≲  ) affinity of the microtubule sub-units for stress granule interfaces. As microtubules polymerize, their interfacial affinity increases, providing sufficient adhesion to deform droplets and/or the network. Our work suggests that proteins and other objects in the cell have a non-specific affinity for droplet interfaces that increases with the contact area and becomes most apparent when they have no preference for the interior of a droplet over the rest of the cytoplasm. We validate this basic physical phenomenon in vitro through the interaction of a simple protein-RNA condensate with microtubules.

Non-specific adhesive forces between filaments and membraneless organelles.

[This corrects the article DOI: 10.1038/s41567-022-01537-8.].

Erratum: Publisher Correction: Non-specific adhesive forces between filaments and membraneless organelles.

Interaction of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) with specific single-stranded RNA and its relation to liquid-liquid phase separation (LLPS) were studied in vitro by magnetic resonance based on site-directed spin labelling. An ensemble model of dispersed hnRNP A1 in the absence of RNA was derived from distance distributions between spin labelled sites and small angle X-ray scattering. This model revealed a compact state of the low-complexity domain and its interaction with the RNA recognition motifs. Paramagnetic relaxation enhancement NMR spectroscopy confirmed this interaction. Addition of RNA to dispersed hnRNP A1 induced liquid-droplet formation. Such LLPS depended on RNA concentration and sequence, with continuous wave EPR spectroscopy showing an influence of RNA point mutations on local protein dynamics. We propose that an interplay of sequence-specific RNA binding and LLPS contributes to regulation of specific RNA segregation during stress response.

Phase Separation of Heterogeneous Nuclear Ribonucleoprotein A1 upon Specific RNA-Binding Observed by Magnetic Resonance.

Solutions of some proteins phase separate into a condensed state of high protein concentration and a dispersed state of low concentration. Such behavior is observed in living cells for a number of RNA-binding proteins that feature intrinsically disordered domains. It is relevant for cell function via the formation of membraneless organelles and transcriptional condensates. On a basic level, the process can be studied in vitro on protein domains that are necessary and sufficient for liquid-liquid phase separation (LLPS). We have performed distance distribution measurements by electron paramagnetic resonance for 13 sections in an N-terminal domain (NTD) construct of the protein fused in sarcoma (FUS), consisting of the QGSY-rich domain and the RGG1 domain, in the denatured, dispersed, and condensed state. Using 10 distance distribution restraints for ensemble modeling and three such restraints for model validation, we have found that FUS NTD behaves as a random-coil polymer under good-solvent conditions in both the dispersed and condensed state. Conformation distribution in the biomolecular condensate is virtually indistinguishable from the one in an unrestrained ensemble, with the latter one being based on only residue-specific Ramachandran angle distributions. Over its whole length, FUS NTD is slightly more compact in the condensed than in the dispersed state, which is in line with the theory for random coils in good solvent proposed by de Gennes, Daoud, and Jannink. The estimated concentration in the condensate exceeds the overlap concentration resulting from this theory. The QGSY-rich domain is slightly more extended, slightly more hydrated, and has slightly higher propensity for LLPS than the RGG1 domain. Our results support previous suggestions that LLPS of FUS is driven by multiple transient nonspecific hydrogen bonding and π-sp interactions.

Ensemble structure of the N-terminal domain (1-267) of FUS in a biomolecular condensate.

Many biomolecular condensates are enriched in and depend on RNAs and RNA binding proteins (RBPs). So far, only a few studies have addressed the characterization of the intermolecular interactions responsible for liquid-liquid phase separation (LLPS) and the impact of condensation on RBPs and RNAs. Here, we present an approach to study protein-RNA interactions inside biomolecular condensates by applying cross-linking of isotope labeled RNA and tandem mass spectrometry to phase-separating systems (LLPS-CLIR-MS). LLPS-CLIR-MS enables the characterization of intermolecular interactions present within biomolecular condensates at residue-specific resolution and allows a comparison with the same complexes in the dispersed phase. We observe that sequence-specific RBP-RNA interactions present in the dispersed phase are generally maintained inside condensates. In addition, LLPS-CLIR-MS identifies structural alterations at the protein-RNA interfaces, including additional unspecific contacts in the condensed phase. Our approach offers a procedure to derive structural information of protein-RNA complexes within biomolecular condensates that could be critical for integrative structural modeling of ribonucleoproteins (RNPs) in this form.

Specific protein-RNA interactions are mostly preserved in biomolecular condensates.

Phase transitions are important to understand cell dynamics, and the maturation of liquid droplets is relevant to neurodegenerative disorders. We combined NMR and Raman spectroscopies with microscopy to follow, over a period of days to months, droplet maturation of the protein fused in sarcoma (FUS). Our study reveals that the surface of the droplets plays a critical role in this process, while RNA binding prevents it. The maturation kinetics are faster in an agarose-stabilized biphasic sample compared with a monophasic condensed sample, owing to the larger surface-to-volume ratio. In addition, Raman spectroscopy reports structural differences upon maturation between the inside and the surface of droplets, which is comprised of β-sheet content, as revealed by solid-state NMR. In agreement with these observations, a solid crust-like shell is observed at the surface using microaspiration. Ultimately, matured droplets were converted into fibrils involving the prion-like domain as well as the first RGG motif.

A solid beta-sheet structure is formed at the surface of FUS droplets during aging.

Liquid-liquid phase separation (LLPS) phenomenon plays a vital role in multiple cell biology processes, providing a mechanism to concentrate biomolecules and promote cellular reactions locally. Despite its significance in biology, there is a lack of conventional techniques suitable for studying biphasic samples in their biologically relevant form. Here, we present a label-free and non-invasive approach to characterize biomolecular condensates termed LLPS REstricted DIFusion of INvisible speciEs (REDIFINE). Relying on diffusion NMR measurements, REDIFINE exploits the exchange dynamics between molecules in the condensed and dispersed phases to determine not only diffusion constants and the fractions in both phases but also the average radius of the condensed droplets and the exchange rate between the phases. Observing proteins, RNAs, water, as well as small molecules, and even assessing the concentrations of biomolecules in both phases, REDIFINE analysis allows a rapid biophysical characterization of multicomponent condensates which is important to understand their functional roles. In comparing multiple systems, REDIFINE reveals that folded RNA-binding proteins form smaller and more dynamic droplets compared to the disordered ones.

LLPS REDIFINE allows the biophysical characterization of multicomponent condensates without tags or labels.

Biomolecular condensates, formed in the process of liquid-liquid phase separation (LLPS), play key roles in RNA metabolism and cellular organization. These dynamic assemblies often contain several components, such as proteins and RNAs. Experimental methods to study biological condensates commonly involve fluorophore-labeling of various droplet components, which may affect phase separation behavior and are thus not able to probe biomolecules in their chemically native state. Here, we introduce a noninvasive, multiplexed NMR approach that enables selective observation of multiple components in hydrogel-stabilized biphasic condensates without using tags. The introduced multiplexing filter combines enhanced spin-spin cross-relaxation in the condensed phase with diffusion and isotope filters to resolve signals from distinct protein pools from both the dilute and condensed phases. We demonstrate the robust performance of this 1D H NMR experiment using biphasic samples containing condensates of FUS N-terminal domain, FUS full-length, and the multicomponent condensate formed by two intrinsically disordered regions of hnRNPC1 protein with highly overlapping resonances. Integrating the multiplexing filter into multidimensional experiments enables site-specific structural and dynamic insights from diverse protein populations, extending NMR applications in LLPS.

ArticleTotal : 2	Year
Publication title Cited by 2	2026
Publication title	2026

Article	Year
Surface tensiometry of phase separated protein and polymer droplets by the sessile drop method. Soft matter\| PubMed ID: 33367441	2021
NMR and EPR reveal a compaction of the RNA-binding protein FUS upon droplet formation. Nature chemical biology\| PubMed ID: 33686294	2021
Characterization of Weak Protein Domain Structure by Spin-Label Distance Distributions. Frontiers in molecular biosciences\| PubMed ID: 33912586	2021
Structural biology of RNA-binding proteins in the context of phase separation: What NMR and EPR can bring? Current opinion in structural biology\| PubMed ID: 34371262	2021
Non-specific adhesive forces between filaments and membraneless organelles. Nature physics\| PubMed ID: 35582428	2022
Erratum: Publisher Correction: Non-specific adhesive forces between filaments and membraneless organelles. Nature physics\| PubMed ID: 35583413	2022
Phase Separation of Heterogeneous Nuclear Ribonucleoprotein A1 upon Specific RNA-Binding Observed by Magnetic Resonance. Angewandte Chemie (International ed. in English)\| PubMed ID: 35866309	2022
Ensemble structure of the N-terminal domain (1-267) of FUS in a biomolecular condensate. Biophysical journal\| PubMed ID: 38279531	2024
Specific protein-RNA interactions are mostly preserved in biomolecular condensates. Science advances\| PubMed ID: 38446881	2024
A solid beta-sheet structure is formed at the surface of FUS droplets during aging. Nature chemical biology\| PubMed ID: 38467846	2024
LLPS REDIFINE allows the biophysical characterization of multicomponent condensates without tags or labels. Nature communications\| PubMed ID: 40389460	2025
Multiplexed Analysis of Multicomponent Biomolecular Condensates without Any Tag. Journal of the American Chemical Society\| PubMed ID: 41360394	2025

Leonidas Emmanouilidis

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