JACS Au

Biomolecular condensates formed via phase separation play critical roles in regulating cellular processes, and their dysregulation is increasingly associated with neurodegeneration. A key challenge in understanding condensate pathology lies in distinguishing dynamic condensates from neurotoxic solid aggregates, the formation of which can, in some systems, occur within existing condensates. Yet current detection techniques often lack the sensitivity and resolution to directly monitor these assemblies and their transitions in dynamic and complex environments. Here, we combined fluorescent probes, based on molecular rotors, with fluorescence lifetime imaging microscopy to quantify changes in protein dynamics during phase separation-associated aggregate formation. Molecular rotors respond to local viscosity changes, which in our study allowed discrimination between fluid condensates and rigid amyloids based on rotors' fluorescence lifetime. Using -synuclein, a hallmark protein in Parkinson's disease, as a model system, we demonstrate real-time visualization of phase separation and condensate maturation within a unified experimental framework. Our method preserves protein integrity and provides quantitative, spatially resolved insights into structural transitions. This technology bridges a critical gap in the study of pathological protein aggregation and offers a powerful platform for mechanistic studies, biomarker discovery, and drug discovery in neurodegenerative disease research.

Organofluorides are ubiquitous in modern society, but their persistence poses significant environmental and health risks. Fluoroacetate dehalogenases (FAcDs) are promising candidates for the bioremediation of polyfluorinated substances, as they can hydrolyze C-F bonds efficiently under mild conditions. However, their activity toward gem-difluorinated carboxylic acids, such as 2,2-difluoroacetate (F2A), is typically poor, with rates orders of magnitude lower than for monofluorinated substrates (e.g. fluoroacetate). Here, we combine kinetic studies, mass spectrometric and biochemical analyses, and computational modeling to pinpoint the F2A-induced isomerization of the catalytic aspartate (Asp105) as a major inactivation pathway that severely limits sustained F2A turnover. To overcome this inhibition mechanism, we discovered that a highly conserved histidine (His104) acts as a gatekeeper for sustained F2A activity. Substituting His104 with the noncanonical N3-methylhistidine increased activity 14-fold, while replacement with asparagine boosted activity almost 50-fold, yielding a potent F2A defluorinase with an initial degradation rate of 0.3 s-1. Computational studies elucidated that these substitutions reshape the conformational landscape of Asp105, suppressing isoAsp-promoting conformations while preserving catalytically productive poses for F2A conversion. Notably, transplanting the His-to-Asn substitution into two homologous FAcDs drastically reduced their activity, highlighting H1 as a uniquely suitable scaffold for engineering promiscuous F2A conversion. Overall, this study demonstrates that targeting mechanistic intricacies can significantly enhance FAcD performance on F2A, providing a foundation for further engineering of H1 to degrade polyfluorinated pollutants.

Rational design of covalent protein-labeling reagents in complex biological environments requires a molecular-level understanding of how the protein microenvironment governs chemical reactivity; yet, such mechanistic details remain inaccessible to experimental methods alone. In living neurons, Ligand-Directed Acyl Imidazole (LDAI) chemistry has been used to label AMPA receptors as a traceless, affinity-based protein labeling method. Although LDAI labeling reagents have been optimized in the lab, the atomic details of their interactions with the protein and the underlying mechanism remain elusive. In this work, we combined Quantum Mechanical (QM) calculations and molecular dynamics (MD) simulations to propose a detailed reaction mechanism for AMPAR labeling by LDAI reagents and to clarify how the protein microenvironment governs reactivity. Although Lys residues are usually protonated at physiological pH and therefore less nucleophilic in water, our QM results show that Lys labeling is energetically more favorable than competing reactions with Ser or water. MD simulations reveal that PFQX ---the LDAI reagent precursor--- binds dynamically to the GluA2 AMPAR as an antagonist, inducing conformational changes that reshape the local environment of the acyl imidazole (AI) warhead, underscoring that ligand identity strongly affects labeling outcomes. We also identified intra and intermolecular hydrogen bond networks that may contribute to further immobilize and pre-organize the LDAI reagent. Moreover, the probe's chemical nature shapes its interactions with the Ligand Binding Domain (LBD), offering a plausible rationale for the previously experimentally observed ligand-dependent fluorescent response. Taken together, our results establish design principles for exploiting the reagent geometry and binding pocket hydrogen-bonding networks for the rational design of LDAI reagents.

Heavy water (D2O) is widely used in biomolecular spectroscopy and imaging, often under the assumption that it is an inert replacement for H2O. However, D2O differs subtly in hydrogen-bonding, viscosity, and dielectric properties, which can alter biomolecular interactions and self-assembly. Here, we test how solvent isotope substitution modulates protein/peptide phase separation and amyloid formation in multiple intrinsically disordered systems. Using turbidity-based phase diagrams and microscopy, we quantify how D2O shifts protein-RNA complex coacervation boundaries and alters condensate morphology. Droplet recovery measurements indicate significant solvent-dependent changes in condensate material properties. We further evaluate amyloid formation kinetics, in the presence or absence of a cofactor, supported by orthogonal structural characterization, and assess the functional consequences of tau fibrils using a tau biosensor seeding assay with explicitly defined seed delivery conditions. Together, these results show that D2O can systematically bias liquid-liquid phase separation and aggregation readouts and should be treated as an active experimental variable rather than a neutral solvent substitute.

Protein phosphatase 2A containing the B56{delta} regulatory subunit (PP2A-B56{delta}) is a critical signaling enzyme whose dysregulation is associated with cancer, neurodegenerative disorders, and Jordan's syndrome, a severe intellectual disability disorder caused by mutations in B56{delta}. Unlike other PP2A holoenzymes, PP2A-B56{delta} is regulated through a unique dual autoinhibition mechanism in which the N- and C-arms occlude the catalytic site while a substrate-mimicking short linear motif (SLiM) blocks the substrate-binding pocket. Although disease-associated mutations have been shown to alter enzyme activity, the molecular mechanism underlying activation of PP2A-B56{delta} and the effects of pathogenic mutations remain poorly understood. Here, we combined cryo-electron microscopy (cryo-EM), enhanced-sampling molecular dynamics (MD) simulations, Markov state model (MSM) construction, and transition-state analysis using Transition State identification via Dispersion and vAriational principle Regularized neural networks (TS-DAR) to characterize the conformational landscape of the disease variant E198K. Our cryo-EM analysis identified two distinct structures of E198K: an inactive closed-form with the N/C-arms resolved and an active loose-form in which the N/C-arms become highly flexible and could not be fully resolved. These structures therefore established that activation is governed by conformational changes of the N/C-arms but did not reveal the underlying mechanism. Starting from the inactive closed-form, we generated over 1,600 trajectories with an average length of 1,260 ns combined for E198K and wild-type (WT) PP2A-B56{delta}. TS-DAR identified four metastable states and two major activation pathways connecting inactive and active conformations. We found that activation occurs through progressive loosening of the N/C-arm interface while maintaining the overall holoenzyme architecture, rather than a complete opening of the interface. This mechanism exposes both the catalytic site and substrate-binding pocket. Comparison of E198K and WT revealed that the disease-associated mutation shifts the conformational equilibrium toward active states while leaving the transition-state ensemble largely unchanged. Mechanistically, E198K disrupts a salt-bridge network and weakens interactions between the internal loop and the C-arm that normally stabilize active-site occlusion. The resulting increase in C-arm mobility promotes active-site exposure and explains the elevated catalytic activity of the mutant. Together, these findings establish a previously uncharacterized activation mechanism for PP2A-B56{delta} and provide an atomic-level explanation for how the pathogenic E198K mutation allosterically promotes holoenzyme activation.

Liquid-state Nuclear Magnetic Resonance (NMR) is an invaluable tool to gain atomic-resolution insights onto molecular structure and dynamics. The impact of NMR spectroscopy, however, is curtailed by high costs and hard-to-maintain high-field magnets. While low-field benchtop NMR spectrometers may address the challenge, severe trade-offs in sensitivity and resolution limit the appeal of this avenue. Here, we introduce a set of novel optically enhanced NMR pulse sequences and procedures (hyperpolarization toolkit) to readily generate 13C and 1H nuclear-spin hyperpolarization in situ on benchtop spectrometers. This approach leads to unprecedented sensitivity gains, enabling atomic-resolution conformation-dependent detection of a broad range of aromatic compounds, including clinically relevant metabolites and biomarkers. Neurotransmitters including epinephrine, serotonin and melatonin are detected at nanomolar/micromolar levels by 1D/2D photochemically-enhanced benchtop NMR in buffer and physiological media including human serum. This advance propels benchtop NMR from a predominantly pedagogical tool to a powerful bioanalytical resource for efficient neurotransmitter identification.

Gene regulation by translational riboswitches relies on repeated and reversible conformational switching between ON (apo) and OFF (holo) states. The switching mechanism often involves significant structural rearrangement, and the role of the metal ions in this mechanism is unclear. Using molecular dynamics simulations, we studied the switching transition of a translational riboswitch that regulates the concentration of S-adenosyl methionine (SAM), the `universal methyl donor'. We show that Mg2+ ions are essential for stabilizing the ON-state, and the three-way junction exhibits a `breathing-like' dynamic interconversion between open and closed modes. The riboswitch has a low population of a holo-like READY (R) state with a partially organized SAM-binding pocket prior to ligand binding. Multiple intermediates are observed in the switching landscape, contributing to the gradual stepwise structural transition between the ON and R states, and proceeding through multiple competing pathways. Interestingly, Mg2+ ions influence the dominant pathway for the ON to R state transition, whereas the reverse transition shows a weak dependence. These results provide broad insights into the function of translational riboswitches, which repeatedly and rapidly respond to changes in metabolite concentrations, in contrast to transcriptional riboswitches.

Nuclear magnetic resonance (NMR) spectroscopy provides atomic-resolution insights into biomolecular structure, dynamics and interactions under non-perturbative conditions. However, the widespread applicability of this technique continues to be limited by its intrinsically low sensitivity. This drawback is particularly severe in the case of concentration-limited samples. Low-concentration photochemically induced dynamic nuclear polarization (LC-photo-CIDNP) is an optically enhanced NMR hyperpolarization strategy capable of boosting NMR sensitivity in situ by several orders of magnitude under physiologically relevant environments. Yet, the inherent nature of the LC-photo-CIDNP phenomenon confines the achievable enhancements to only a subset of nuclei, thus limiting the extent of attainable residue-specific information. Here, we overcome this shortcoming by integrating LC-photo-CIDNP with Total Correlation Spectroscopy (TOCSY) to propagate initial hyperpolarization to other nuclei via scalar-coupled spin networks. First, we demonstrate efficient through-bond transfer of LC-photo-CIDNP enhanced signals throughout entire intramolecular atomic frameworks via 1D 1H- and 13C-detected experiments. Second, we extend the approach to a model protein and demonstrate efficient polarization transfer via 1D and 2D 1H-detected 13C-TOCSY LC-photo-CIDNP. Intrinsically photo-CIDNP-inactive nuclei can thus be visualized via 13C-1H correlations within minutes down to 10 M concentration. In all, this work establishes a general strategy to achieve through-bond propagation of LC-photo-CIDNP hyperpolarization. This technology enables gaining residue-specific structural insights on amino acids and proteins at previously unattainable low concentrations.

Mucopolysaccharidosis IIIC (Sanfilippo syndrome type C) is a rare lysosomal storage disorder caused by loss-of-function mutations in HGSNAT, which encodes an enzyme involved in heparan sulfate (HS) degradation, leading to impaired HS catabolism, lysosomal accumulation, and progressive neurodegeneration. Because enzyme replacement therapies have limited penetration across the blood-brain barrier, substrate-reduction therapy represents an alternative therapeutic strategy. Here, N-deacetylase/N-sulfotransferase 1 (NDST1), a key enzyme responsible for HS biosynthesis, was investigated as a potential substrate-reduction target. A structure-based computational pipeline was used to identify and evaluate inhibitors targeting the NDST1 sulfotransferase domain. Approximately 4.1 million drug-like compounds and FDA-approved drugs were screened by molecular docking, followed by pharmacokinetic filtering, molecular dynamics simulations, and MM/PBSA binding free energy calculations. In parallel, peptide binders targeting the same site were generated using diffusion-based protein design and evaluated using molecular dynamics and MM/GBSA analysis. Four chemically distinct small-molecule scaffolds and three peptide candidates were identified as stable binders to the NDST1 active site. The lead small-molecule candidate exhibited a predicted binding free energy of -13.36 {+/-} 5.87 kcal mol-1. These provide a focused set of candidates for further investigation and support the feasibility of targeting NDST1 as a substrate-reduction strategy for MPS IIIC.

G protein-coupled receptors (GPCRs) represent the largest membrane protein family in the human body and the major targets of FDA-approved medications. Unlike some other physiological proteins, GPCRs are highly plastic and often exist as collections of conformational ensembles in the environment to exert their function. Therefore, understanding how a GPCR adopts different conformational ensembles in response to ligands and signaling partners is essential for drug development. 19F-NMR has emerged as one of the most promising tools in this regard; however, a well-established 19F-labeling system is still lacking, including an effective 19F probe that meets both environmental susceptibility and NMR sensitivity requirements, even though progress has been made dramatically in recent years. Thus, this manuscript reports a novel conformational probe (4-(bromomethyl)-ethoxy-2-(trifluoromethyl)benzene, BTMB) that offers paramount benefits for future GPCR conformational dynamics studies. To rationalize the mechanism behind BTMB's superiority for future probe development, we performed MD simulations, referenced to the current state-of-the-art probe, BTFMA. Our study suggests that the spatial proximity of aromatic residues significantly influences the chemical shift dispersion and linewidths of related 19F-probe-profiled conformational states, as well as the rigidity and compactness of -CF3 in the target protein.

Variable-temperature electrospray ionization native mass spectrometry (vT-ESI nMS) provides a means to determine how temperature-dependent changes in protein and solution dynamics influence individual ligand-binding reactions within multiligand protein complexes. Here, the GroEL single-ring mutant (SR1), which binds up to seven nucleotides, serves as a model system due to its sensitivity to solution conditions. Native MS resolves individual ligand-bound populations, while vT-ESI extends these measurements across temperature (3-43 {degrees}C), enabling determination of binding affinities and the associated Gibbs free energy, enthalpy, entropy, heat capacity changes ({Delta}Cp), and enthalpy-entropy compensation (EEC) for each binding reaction. Temperature-dependent changes in van,t Hoff curvature produce distinct {Delta}Cp profiles, indicating changes in the dynamics governing ligand binding that may reflect contributions from protein conformational and protonation microstates. Temperature-dependent shifts in average charge state, consistent with changes in solvent-accessible surface area, indicate corresponding changes in protein conformation. Binding is predominantly enthalpy-driven below ~23 {degrees}C, with increasing entropic contributions at higher temperatures, while the seventh ADP binding reaction exhibits a distinct EEC profile. Comparison of the seventh ADP binding reaction in H2O and D2O reveals pronounced differences in van,t Hoff curvature,{Delta}Cp, and EEC, demonstrating that changes in the hydration environment alter the thermodynamic response. Collectively, these measurements show that temperature-dependent changes in protein and solution dynamics alter the distributions and thermodynamics of individual ligand-bound states and demonstrate the utility of vT-ESI nMS for resolving these effects in complex multiligand systems.

Nanoplastic contamination is increasingly recognized as a biochemical stressor, yet the molecular determinants governing how polymer fragment size dictates protein-level interactions remain poorly understood. Here, we combine molecular docking and all-atom molecular dynamics simulations to characterize the interaction of polyethylene (PE) oligomers of increasing length (n = 3, 5, 7, and 14 repeat units) with human catalase, a tetrameric heme enzyme central to cellular antioxidant defense. Single-chain simulations reveal that short PE fragments (3-, 5-, and 7-mer) migrate from an initial domain-connector pose toward a heme-proximal, previously characterized inhibitor-binding site, whereas the 14-mer remains kinetically confined to a peripheral surface site, implicating short-chain PE as a potential catalase inhibitor. In multichain corona systems, backbone dynamics show a non-monotonic, chain-length-dependent destabilization, with the 3- and 5-mer inducing the greatest structural perturbation and the 14-mer best preserving native-like dynamics despite forming an extensive, percolating surface network across all four subunits. MM-PBSA free energy decomposition identifies van der Waals interactions as the dominant thermodynamic driver of binding, while electrostatic surface mapping indicates that cationic surface patches instead govern long-range spatial targeting of PE chains. These findings establish a framework linking nanoplastic fragment size to protein-level toxicity, with direct implications for assessing the biochemical risk of environmentally weathered polyethylene.

Chemically similar post-translational modifications can mediate distinct biological functions, but how proteins distinguish between them remains unclear. Sulfotyrosine (sTyr) and phosphotyrosine (pTyr) exemplify this problem because they have similar sizes, local geometries, and electrostatic properties but function in different biological contexts. Here, we used the monoclonal antibody PSG2, which recognizes sTyr independently of the surrounding peptide sequence, to examine how a protein distinguishes these modifications. The crystal structure of PSG2 bound to an sTyr-containing peptide revealed a deep electropositive pocket with no modeled water molecules in direct contact with the sulfate group. Gas-phase density functional theory calculations favored pTyr over sTyr, showing that direct protein-ligand interactions alone are insufficient to explain PSG2 selectivity. Explicit first-shell hydration calculations showed that pTyr has a larger desolvation penalty than sTyr, and accounting for this difference reversed the calculated energetic order. Isothermal titration calorimetry showed favorable enthalpic and entropic contributions to sTyr binding, whereas no detectable heat signal was observed for pTyr. These results show that PSG2 distinguishes sTyr from pTyr through the balance between direct protein-ligand interactions and ligand desolvation.

Transition-metal ion Forster resonance energy transfer (tmFRET) measures short-range distances and conformational changes in proteins, but its distance sensitivity depends on the donor-acceptor pair. Here, we establish the metal-chelating noncanonical amino acid bipyridylalanine (bpyA) as a flexible, genetically encoded acceptor platform whose Forster distance (R0) can be tuned without changing protein labeling. Cu2+, Cu2+ complexed with nitrilotriacetic acid (NTA), and Fe2+ form distinct acceptors at the same bpyA site, allowing distance sensitivity to be adjusted by changing the solution conditions. We incorporated bpyA at two sites in maltose binding protein (MBP) and labeled nearby engineered cysteines with the donor fluorophore BODIPY. The resulting [Cu(bpyA)]2+, [CuNTA(bpyA)]2+, and [Fe(bpyA)]2+ acceptors provided R0 values ranging from 13.9 to 21.2 [A]. Analysis of time-resolved tmFRET data recovered donor-acceptor distance distributions and maltose-dependent changes consistent with structural predictions. At the 237/295 site pair, both copper and iron acceptors resolved the maltose-dependent decrease in distance. At the 309/322 site pair, [Cu(bpyA)]2+ did not appreciably resolve the distance increase, whereas adding NTA shifted R0 toward shorter distances and enabled detection of an increase from 12.1 to 14.4 [A]. These results demonstrate that a single bpyA-labeled protein preparation supports multiple tmFRET acceptors, providing a cysteine-independent acceptor-labeling strategy with adjustable distance sensitivity.

Sulfur assimilation fuels bacterial growth by supplying the reduced sulfur required for the biosynthesis of sulfur-containing biomolecules. Adenosine 5'-phosphosulfate reductase (APSR) catalyzes the first reductive step of this pathway, converting adenosine 5'-phosphosulfate (APS) to adenosine monophosphate. This reaction proceeds through nucleophilic attack by catalytic C256, located in the flexible C-terminal tail, on the sulfur atom of APS, forming a thiosulfonate enzyme intermediate. Here, we investigate this reaction in APSR from Pseudomonas aeruginosa, an opportunistic pathogen associated with severe infections, particularly in patients with cystic fibrosis. APSR contains an iron-sulfur [4Fe-4S] cluster, which participates in redox steps of the reaction. Here, we show that the redox state of the iron-sulfur cluster also controls the catalytic step. Multiscale molecular simulations investigate how oxidized and reduced cluster states affect APS binding, active site organization, and the nucleophilic attack step. Molecular dynamics (MD) simulations show that the oxidized [4Fe-4S]2+; cluster stabilizes substrate interactions and the conformation of the C-terminus, facilitating a catalytically productive orientation of C256. The activation barrier of 17.7 {+/-} 1.7 kcal mol-1 from quantum mechanics/molecular mechanics (QM/MM) umbrella sampling MD simulations at the B3LYP-D3(BJ)/6-31G(d) level of theory is in good agreement with the experimental kinetics. The redox state of the iron-sulfur cluster shows its role in modulating the conformation of conserved K144, which is important for transition state stabilization in the nucleophilic attack. These findings illuminate the mechanism of this P. aeruginosa target and provide broader insight into the roles of iron-sulfur clusters in controlling enzyme reactivity.

Reactive metal-oxygen intermediates are central to metalloenzyme catalysis, yet their low populations and short lifetimes make vibrational characterization under aqueous reaction conditions challenging. Here, we establish shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) as a powerful vibrational approach for visualizing reaction-associated metal-ligand signatures of reactive metalloenzyme states. Across the heme and non-heme iron enzymes TauD, VioC, CYP119, and TqaLNc, isotope-sensitive features provide vibrational information on reactive coordination states. In TauD and VioC, isotope-sensitive features provide vibrational characterization of Fe(IV)=O intermediates. In CYP119, an isotope-sensitive Fe(IV)=O vibration is assigned to P450 Compound I generated by m-CPBA, including under lauric-acid turnover conditions. Most notably, in TqaLNc, combined 18O- and 15N-sensitive vibrational responses support formation of the proposed (R)HN-Fe(IV)=O coordination state and the dynamic-coordination mechanism underlying selective C-N bond formation. These results demonstrate that SHINERS can serve as a powerful technique for the in situ vibrational visualization of reactive intermediates and their coordination states in metalloenzyme catalysis.

Investigating the conformational dynamics of intrinsically disordered proteins (IDPs) is essential to understanding how their structural heterogeneity underlies function and how their dysregulation contributes to diseases. Here, we utilized an MspA nanopore-based approach for studying the conformational dynamics and interactions of IDPs at the single-molecule level. The platform was demonstrated using the intrinsically disordered transactivation domain of tumor suppressor p53 (p53-TAD), one of the important proteins in cancer biology. We showed that MspA can stably capture p53-TAD and resolve up to six distinct current states with frequent interconversions, revealing a rich conformational landscape. The nanopore also detected the effect of a cancer-associated double mutational variant, N29K/N30D. Combining experiments with steered molecular dynamics simulations, we showed that the mutant sampled compact conformational states more frequently than wild type, consistent with previous NMR studies. Importantly, the MspA platform enabled direct monitoring of E3 ligase MDM2 binding to p53-TAD and resolved how this interaction is inhibited by anti-cancer compound epigallocatechin gallate (EGCG). Notably, EGCG stabilizes one of the six states sampled by p53-TAD, providing a mechanistic explanation for its inhibitory effect. Together, these findings demonstrate the promise of the nanopore platform for label-free monitoring of IDP conformational dynamics, modulation, binding and inhibition at single-molecule resolution.

Allosteric and cryptic binding sites provide unique opportunities in drug discovery for achieving selectivity, reducing toxicity and unlocking challenging therapeutical targets. Yet, cryptic pockets are particularly elusive in that they are difficult to capture with experimental methodologies, and therefore often remain undetected. Computational approaches for flexible cryptic site detection traditionally involve extensive unbiased molecular dynamics simulations, or more efficient but computationally still highly demanding biased MD alternatives that make use of enhanced sampling along low-frequency motions of the protein structure. Here we utilize a new variant of the recently introduced and orders of magnitude faster, MD-free COVIB algorithm to identify cryptic pockets efficiently in two challenging targets, the KRAS oncoprotein and the N-terminal domain of the STAT5B transcription factor. Using experimental and AI-modelled structures, respectively, we identify numerous cryptic sites, some of which have not yet been reported. The sites are prioritized and validated with our pharmacophore optimized photoaffinity-tagged fragment library via MS-based peptide mapping and NMR spectroscopy. Besides identifying new, targetable regions on the surface of these important oncotargets, we have established a protocol combining COVIB-based cryptic pocket detection and photoaffinity-based experimental peptide mapping as a robust approach for exploring unique and tractable cryptic pockets.

RNA interactions are a key contributor to the formation and disassembly of intracellular protein condensates. Although some proteins utilize specific RNA-binding domains, these processes can also be mediated by charge interactions with intrinsically disordered regions. Due to the dynamic nature of these systems, investigating the underlying specificity and stoichiometry remains challenging. Here, we demonstrate that single-molecule mass measurements with mass photometry can capture RNA-protein interactions in phase-separated protein systems. Using the approach to investigate RNA-mediated phase shifts of tau condensates, we find that increasing the RNA concentration, which promotes phase re-entry, results in RNA-mediated tau multimerization, where each tau monomer binds a linear RNA sequence of approximately 30 nucleotides. Solution NMR and native mass spectrometry confirm the formation of stable complexes between RNA and the basic proline-rich and repeat domains of tau, which have a net charge of -29. Our findings demonstrate that mass photometry can distinguish between charge neutralization, which drives coacervation, and complex formation, which mediates phase re-entry, making it a highly complementary tool for the study of RNA-mediated phase separation.

Fluorescent base analogues have proven to be valuable probes of RNA structure. Pyrrolocytosine (pC), an analogue of cytosine, exhibits highly environment-sensitive fluorescence within RNA and is strongly quenched in hydrogen-bonded contexts. In this work, we incorporated pC into the aptamer domain of the B. subtilis guanine riboswitch to determine if this quenching mechanism could allow for sensing of RNA-small molecule interactions. We utilized complementary fluorescence spectroscopy methods to under-stand the functional impacts and ligand-sensitivity of pC substitutions distal to and within the binding pocket. We found that pC substitution within a static structural region exhibits the expected ligand-independent fluorescence properties. When placed in the ligand binding pocket, pC exhibits a moderately decreased affinity for the ligands guanine (G) and hypoxanthine (Hx) while maintaining high specificity for G. We found that pC behaves as a turn-off sensor, with distinct changes in fluorescence upon G or Hx binding. These differences are dependent on the number of hydrogen bonds formed with pC and are resolvable in both time-dependent and steady-state fluorescence measurements. Our results show that pC can be used to study RNA-small molecule interactions and can discriminate between similar ligands even when they have identical binding modes.