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

The ongoing pandemic of the respiratory disease COVID-19 is caused by the SARS-CoV-2 (SCoV2) virus. SCoV2 is a member of the Betacoronavirus genus. The 30 kb positive sense, single stranded RNA genome of SCoV2 features 5'- and 3'-genomic ends that are highly conserved among Betacoronaviruses. These genomic ends contain structured cis-acting RNA elements, which are involved in the regulation of viral replication and translation. Structural information about these potential antiviral drug targets supports the development of novel classes of therapeutics against COVID-19. The highly conserved branched stem-loop 5 (SL5) found within the 5'-untranslated region (5'-UTR) consists of a basal stem and three stem-loops, namely SL5a, SL5b and SL5c. Both, SL5a and SL5b feature a 5'-UUUCGU-3' hexaloop that is also found among Alphacoronaviruses. Here, we report the extensive H, C and N resonance assignment of the 37 nucleotides (nts) long sequence spanning SL5b and SL5c (SL5b + c), as basis for further in-depth structural studies by solution NMR spectroscopy.

H, C and N chemical shift assignment of the stem-loops 5b + c from the 5'-UTR of SARS-CoV-2.

The pandemic caused by SARS-CoV-2 has called for concerted efforts to generate new insights into the biology of betacoronaviruses to inform drug screening and development. Here, we establish a workflow to determine the RNA recognition and druggability of the nucleocapsid N-protein of SARS-CoV-2, a highly abundant protein crucial for the viral life cycle. We use a synergistic method that combines NMR spectroscopy and protein-RNA cross-linking coupled to mass spectrometry to quickly determine the RNA binding of two RNA recognition domains of the N-protein. Finally, we explore the druggability of these domains by performing an NMR fragment screening. This workflow identified small molecule chemotypes that bind to RNA binding interfaces and that have promising properties for further fragment expansion and drug development.

A hybrid structure determination approach to investigate the druggability of the nucleocapsid protein of SARS-CoV-2.

INEPT-based experiments are widely used for  H→ N transfers, but often fail when involving labile protons due to solvent exchanges. J-based cross polarization (CP) strategies offer a more efficient alternative to perform such transfers, particularly when leveraging the H  H exchange process to boost the  H→ N transfer process. This leveraging, however, demands the simultaneous spin-locking of both H and H protons by a strong  H RF field, while fulfilling the γ B =γ B Hartmann-Hahn matching condition. Given the low value of γ /γ , however, these demands are often incompatible-particularly when experiments are executed by the power-limited cryogenic probes used in contemporary high field NMR. The present manuscript discusses CP alternatives that can alleviate this limitation, and evaluates their performance on urea, amino acids, and intrinsically disordered proteins. These alternatives include new CP variants based on frequency-swept and phase-modulated pulses, designed to simultaneously fulfill the aforementioned conflicting conditions. Their performances vis-à-vis current options are theoretically analyzed with Liouville-space simulations, and experimentally tested with double and triple resonance transfer experiments.

Cross-Polarization Schemes for Improved Heteronuclear Transfers Involving Labile Protons in Biomolecular Solution NMR.

RNA-binding proteins (RBPs) are crucial regulators of gene expression, often composed of defined domains interspersed with flexible, intrinsically disordered regions. Determining the structure of ribonucleoprotein (RNP) complexes involving such RBPs necessitates integrative structural modeling due to their lack of a single stable state. In this study, we integrate magnetic resonance, mass spectrometry, and small-angle scattering data to determine the solution structure of the polypyrimidine-tract binding protein 1 (PTBP1/hnRNP I) bound to an RNA fragment from the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). This binding, essential for enhancing the translation of viral RNA, leads to a complex structure that demonstrates RNA and protein compaction, while maintaining pronounced conformational flexibility. Acting as an RNA chaperone, PTBP1 orchestrates the IRES RNA into a few distinct conformations, exposing the RNA stems outward. This conformational diversity is likely common among RNP structures and functionally important. Our approach enables atomic-level characterization of heterogeneous RNP structures.

Integrative solution structure of PTBP1-IRES complex reveals strong compaction and ordering with residual conformational flexibility.

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.

The SARS-CoV-2 nucleocapsid (N) protein is crucial for virus replication and genome packaging. N protein forms biomolecular condensates both in vitro and in vivo in a process known as liquid-liquid phase separation (LLPS), but the exact factors regulating LLPS of N protein are not fully understood. Here, we show that pH and buffer choice have a profound impact on LLPS of N protein. The degree of phase separation is highly dependent on the pH of the solution, which is correlated with histidine protonation in N protein. Specifically, we demonstrate that protonation of H356 is essential for LLPS in phosphate buffer. Moreover, electrostatic interactions of buffer molecules with specific amino acid residues are able to alter the net charge of N protein, thus influencing its ability to undergo phase separation in the presence of RNA. Overall, these findings reveal that even subtle changes in amino acid protonation or surface charge caused by the pH and buffer system can strongly influence the LLPS behavior, and point to electrostatic interactions as the main driving forces of N protein phase separation. Further, our findings emphasize the importance of these experimental parameters when studying phase separation of biomolecules, especially in the context of viral infections where the intracellular milieu undergoes drastic changes and intracellular pH normally decreases.

Buffer choice and pH strongly influence phase separation of SARS-CoV-2 nucleocapsid with RNA.

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.

RNA condensation is implicated in the formation of neurotoxic RNA foci in cells affected by genomic expansions of trinucleotide or hexanucleotide repeats. However, the biophysical properties of repeat-expansion RNA condensates are poorly understood. Using CAG repeat-expansion RNA as a model system, we show that these RNA condensates cannot be observed with conventional nuclear magnetic resonance techniques. Therefore, we developed a nuclear magnetic resonance approach, based on water-detected semi-solid magnetization transfer, to detect and characterize RNA condensates in vitro. Our method, termed condensate detection by semi-solid magnetization transfer (CONDENSE-MT), is broadly applicable, highly sensitive and does not require direct observation of the biomolecules of interest. Using CONDENSE-MT, we could obtain dynamic information about RNA condensates, such as the relative amount and the tumbling rate of condensed RNA, the proton-solvent exchange kinetics and the amount of water molecules transiently bound in the condensate. We find that phase separation dramatically decreases molecular tumbling and is driven by heterotypic interactions between RNA and Mg. We further show that increasing CAG repeats decreases condensate hydration.

Water-detected NMR allows dynamic observations of repeat-expansion RNA condensates.

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
H, C and N chemical shift assignment of the stem-loops 5b + c from the 5'-UTR of SARS-CoV-2. Biomolecular NMR assignments\| PubMed ID: 35178672	2022
A hybrid structure determination approach to investigate the druggability of the nucleocapsid protein of SARS-CoV-2. Nucleic acids research\| PubMed ID: 36928389	2023
Cross-Polarization Schemes for Improved Heteronuclear Transfers Involving Labile Protons in Biomolecular Solution NMR. Angewandte Chemie (International ed. in English)\| PubMed ID: 37408374	2023
Integrative solution structure of PTBP1-IRES complex reveals strong compaction and ordering with residual conformational flexibility. Nature communications\| PubMed ID: 37833274	2023
Specific protein-RNA interactions are mostly preserved in biomolecular condensates. Science advances\| PubMed ID: 38446881	2024
Buffer choice and pH strongly influence phase separation of SARS-CoV-2 nucleocapsid with RNA. Molecular biology of the cell\| PubMed ID: 38568799	2024
LLPS REDIFINE allows the biophysical characterization of multicomponent condensates without tags or labels. Nature communications\| PubMed ID: 40389460	2025
Water-detected NMR allows dynamic observations of repeat-expansion RNA condensates. Nature chemistry\| PubMed ID: 41094266	2025
Multiplexed Analysis of Multicomponent Biomolecular Condensates without Any Tag. Journal of the American Chemical Society\| PubMed ID: 41360394	2025

Mihajlo Novakovic

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