JACS Au

Per- and polyfluoroalkyl substances (PFAS) are harmful environmental contaminants that bioaccumulate in human tissues and are linked to adverse health outcomes. While PFAS are known to bind to a variety of lipid binding proteins (LBPs), such as human serum albumin and fatty acid-binding proteins (FABPs), the broader molecular basis for their biological distribution and breadth of protein binding in humans remains unanswered. We hypothesize that some distribution and persistence of PFAS in humans arises from a distributed network of lipid transfer proteins that collectively solubilize and transport these compounds. To support this hypothesis, we investigated the interaction between various PFAS and human sterol carrier protein 2 (SCP2), a promiscuous, structurally distinct LBP with no previously reported binding with PFAS. Using a combination of screening, fluorescence displacement assays, protein structure prediction of PFAS-SCP2 complexes, and NMR experiments, we demonstrate for the first time that SCP2 is a PFAS-binding protein. Our findings establish SCP2 as a new PFAS-interacting protein, providing insights into the residues participating in these interactions and further supporting the hypothesis that PFAS engage with a broad network of LBPs to facilitate their distribution and persistence in the human body.

NSD2 is a histone methyltransferase that modifies lysine 36 in histone H3 (H3K36), playing a central role in chromatin organization and transcriptional regulation. Oncogenic mutations, such as E1099K and T1150A in NSD2, have been associated with hyperactive methylation, but the molecular mechanisms underlying this gain of function remain poorly understood. In this study, we performed all-atom molecular dynamics simulations on models of NSD2 bound to the nucleosome for the wild type (WT), E1099K, T1150A, and the E1099K/T1150A double mutant. Analysis of MD simulations reveals that the global dynamics of the enzymes remain unaltered upon mutations. The time-lagged independent component analysis (tICA) and Markov state modeling uncovered fundamental differences in free-energy landscapes among the variants. The WT NSD2 exhibited energetically and kinetically unfavorable transitions between the macrostates along with extended enzyme-substrate distances. On the other hand, the mutant systems demonstrate reduced SAM-H3K36 distances with modified energy landscapes that facilitate transitions or favor prolonged occupancy of catalytically competent states. Importantly, the mutations reorganize the network of intramolecular contacts around the catalytic site, SAM-binding pocket, and histone-binding interface, optimizing substance engagement geometry. These findings demonstrate that oncogenic mutations achieve hyperactivity through strategic reorganization of conformational dynamics rather than simple destabilization, balancing local flexibility with global stability to enhance catalytic efficiency. Our results provide mechanistic insights into NSD2 dysregulation in cancer and establish a framework to develop allosteric inhibitors that target the enzymes dynamic landscape.

Parkinsons Disease (PD) is characterized by the aggregation of alpha-synuclein (aSyn), a presynaptic protein that transitions from a disordered monomer into beta-sheet rich amyloid fibrils. The precise triggers and mechanisms underlying aSyn misfolding and aggregation remain unclear, hindering the development of effective therapeutics. Monomeric aSyn is an intrinsically disordered protein (IDP) with high conformational flexibility. Local environmental factors, such as ion concentrations, can influence the conformational ensemble of aSyn, impacting its aggregation propensity and resulting in fibril polymorphism. In this study, we explore the impact of physiologically relevant ions, mainly Ca2+ and Na+, on the aggregation kinetics, monomer structural dynamics, and fibril polymorphism of aSyn. Using ThT fluorescence assays, we demonstrate that all ions accelerate aSyn aggregation, with Ca2+ having the most significant effect. Using Heteronuclear Single Quantum Correlation Nuclear Magnetic Resonance (1H-15NHSQC NMR) spectroscopy, we validate the specific binding of Ca2+ ions at the C-terminus, whereas Na+ ions display non-specific interactions along the sequence of aSyn. Small-angle neutron scattering (SANS) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) further reveal that Na+ and Ca2+ induce distinct conformational changes in the aSyn monomer, with Na+ leading to more extended structures and Ca2+ promoting a moderate extension of the protein. Molecular dynamics simulations (MD) corroborate these findings, showing that Na+ ions increase the proteins extension, particularly between the non-amyloid beta component (NAC) region and the C-terminus, whereas Ca2+ ions bias the ensemble towards a more moderately elongated structure. Using MD, we further investigate the local environment and in particular the solvent effect and show the water persistence times in the hydration shell are also increased in the presence of Ca2+ ions, indicating that the aggregation propensity of the monomer is due to a combination of conformational bias of the monomer and solvent mobility. Atomic force microscopy (AFM) of aSyn fibrils formed under these different ionic conditions reveal distinct fibril polymorphs, suggesting that ion-induced conformational biases in the monomer contribute to the diversity of fibril structures. Collectively, these findings underscore the pivotal influence of the local ionic milieu in shaping the structure and aggregation propensity of aSyn, thus offering valuable insights into the molecular underpinnings of PD and potential therapeutic avenues aimed at manipulating aSyn conformational dynamics.

The aggregation of -synuclein (Syn) is a molecular hallmark of Parkinson's disease (PD) and other synucleinopathies. Understanding the molecular mechanisms that determine the aggregation of this protein may thus facilitate the development of disease-modifying therapies. While Syn is most commonly expressed as a 140-residue protein (Syn-140), recent evidence suggests an involvement of alternatively spliced Syn isoforms in disease onset and progression. Here, we report and characterise the interaction between Syn-140 and the aggregation-prone Syn-112 variant, one of the most abundant Syn splice isoforms. We found that amounts as low as 1% of Syn-112 accelerate the nucleation and aggregation of Syn-140. To further investigate this phenomenon, we employed MALDI-MS and NMR spectroscopy, confirming that Syn-140 and Syn-112 monomers interact strongly with one another. Furthermore, to assess the association of Syn-112 with disease pathology, we performed immunohistochemical staining combined with confocal microscopy on PD brain samples. Thereby, we found an increase in the number as well as the area of Syn-112 immunoreactive aggregates compared to healthy controls. These results illustrate how low-abundance Syn splice isoforms can modulate the aggregation landscape of Syn-140 and in turn contribute to the molecular heterogeneity of synucleinopathies.

The rapid rise of antibiotic-resistant bacteria necessitates the search for alternative, unconventional solutions, such as targeting bacterial communication. Signal disruption can be achieved by enzymatic degradation of signaling compounds, reducing the expression of genes responsible for virulence, biofilm formation, and drug resistance while evading common resistance mechanisms. Therefore, enzymes with such activity have considerable potential as antimicrobial agents for medicine, industry, and other areas of life. Here, we designed molecular gates that control the binding site of penicillin G acylase to shift its preference from native substrate to signaling molecules. Using an ensemble-based design, three variants carrying triple-point mutations were proposed and experimentally characterized. Integrated inference from biochemical and computational analyses demonstrated that these three variants had markedly reduced activity towards penicillin and each preferred specific signal molecules of different pathogenic bacteria, exhibiting up to three orders of magnitude shifts in substrate specificity. Curiously, while we could consistently expand the pockets in these mutants, the reactive binding of larger substrates was limited, either by overpromoting or overstabilizing the pocket dynamics. Overall, we demonstrated the designability of this acylase for signal disruption and provided insights into the role of appropriately modulated pocket dynamics for such a function. The improved mutants, the knowledge gained, and the computational workflow developed to prioritize large datasets of promising variants may provide a suitable toolbox for future exploration and design of enzymes tailored to disrupt specific signaling pathways as viable antimicrobial agents.

As an intrinsically disordered protein, monomeric alpha synuclein (aSyn) constantly reconfigures and probes the conformational space. Long-range interactions across the protein maintain its solubility and mediate this dynamic flexibility, but also provide residual structure. Certain conformations lead to aggregation prone and non-aggregation prone intermediates, but identifying these within the dynamic ensemble of monomeric conformations is difficult. Herein, we used the biologically relevant calcium ion to investigate the conformation of monomeric aSyn in relation to its aggregation propensity. By using calcium to perturb the conformational ensemble, we observe differences in structure and intra-molecular dynamics between two aSyn C-terminal variants, D121A and pS129, and the aSyn familial disease mutants, A30P, E46K, H50Q, G51D, A53T and A53E, compared to wild-type (WT) aSyn. We observe that the more exposed the N-terminus and the beginning of the NAC region are, the more aggregation prone monomeric aSyn conformations become. N-terminus exposure occurs upon release of C-terminus interactions when calcium binds, but the level of exposure is specific to the aSyn mutation present. There was no correlation between single charge alterations, calcium affinity, or the number of ions bound on aSyns aggregation propensity, indicating that sequence or post-translation modification (PTM)-specific conformational differences between the N- and C-termini and the specific local environment mediate aggregation propensity instead. Understanding aggregation prone conformations of monomeric aSyn and the environmental conditions they form under will allow us to design new therapeutics targeted to the monomeric protein, to stabilise aSyn in non-aggregation prone conformations, by either preserving long-range interactions between the N- and C-termini or by protecting the N-terminus from exposure.

Post-translational modifications (PTMs) of the tau protein are increasingly recognized as pivotal regulators in the onset and progression of tauopathies, such as Alzheimers disease (AD). To systematically evaluate the structural and functional consequences of specific PTMs, we generated and analyzed seven distinctly modified variants of the tau-K32 construct. These included phosphorylation at Ser202/Thr205, phosphorylation at Ser258/Ser262/Ser356, full phosphorylation at all reported Ser/Thr sites, acetylation at Lys274/Lys281, acetylation at Lys280, full acetylation at all sites, and an unmodified control. Selection of PTM sites was guided by prior experimental literature. By incorporating fully modified tau models, we assessed the global impact of widespread modifications on structural properties and aggregation behavior. Our findings establish a comparative framework for understanding how discrete and cumulative PTMs modulate tau aggregation and provide mechanistic insight into PTM-induced tau dysfunction relevant to neurodegenerative diseases.

Membrane transporters move polar solutes across lipid bilayers to regulate cellular metabolism, signaling, and drug distribution. These proteins operate via an alternating-access mechanism, cycling between extracellular-, intermediate-, and intracellular-facing conformations. The human excitatory amino acid transporter 1 (EAAT1) protects neurons from excitotoxic damage by mediating the uptake of glutamate and aspartate into glial cells. Defects in EAAT1 function result in numerous pathologies, including epilepsy and ataxia, suggesting that positive modulation of these transporters might ameliorate glutamate neurotoxicity. However, developing EAAT1 activators requires understanding the timing of conformational changes, which remain largely unexplored. Here, we establish an experimental platform that combines single-molecule Forster resonance energy transfer (smFRET) to monitor real-time conformational dynamics, single-transporter activity assays to correlate dynamics with function, and cryogenic electron microscopy (cryoEM) to visualize discrete conformations at high resolution. This platform enables detection of [A]ngstrom-scale movements of single transporter molecules in real time, revealing that EAAT1 intersperses rapid conformational dynamics with long pauses. Slow and fast dynamics can be modulated by substrates, membrane composition, and mutations, and are correlated with the enrichment of specific structural states. We leverage this platform to investigate an EAAT1 mutation associated with severe episodic ataxia and show that it inhibits transport by stabilizing a paused cytoplasm-facing conformation. These results identify multimodal dynamics as an intrinsic, regulatable feature of EAAT1 function and, therefore, a potential therapeutic target. Henceforth, our integrated platform will facilitate investigations of other regulatory factors, including the effects of small-molecule and lipid modulators on the transport cycle.

I-motif (iM) DNA structures, formed by cytosine-rich sequences, are increasingly acknowledged for their involvement in gene regulation, maintenance of genomic stability, and their emerging potential as therapeutic targets, particularly in cancer. Despite their biological relevance, the discovery of selective small-molecule probes that can specifically recognize and interact with iM DNA remains an ongoing challenge. In this study, we have used TMPyP4 and screened for its ability to bind various iM DNA constructs, including HRAS1, HRAS2, VEGF, CMYC, CKIT and H-Telo. Structure-activity relationship analyses revealed that specific substitution patterns conferred selectivity towards HRAS2 iM target. Comprehensive spectroscopic investigations, including UV-Vis absorption, steady-state and time-resolved fluorescence, and fluorescence anisotropy, uncovered key photophysical signatures of binding, including significant hypochromic and bathochromic shifts, enhanced fluorescence emission, and prolonged fluorescence lifetimes. Circular dichroism (CD),thermal denaturation (UV-melting) and thermodynamic investigations confirmed that TMPyP4 effectively stabilized the HRAS2 iM structures without disrupting their native topologies. Meanwhile, FT-IR spectroscopy revealed local structural rearrangements upon TMPyP4 binding, offering further evidence of molecular interaction. Collectively, these findings provide valuable insights into the molecular recognition of iM DNA by TMPyP4 and highlight its promise as both selective HRAS2 iM-binding agent and responsive fluorescent probe. This work lays a strong foundation for the development of novel tools for studying iM structures in biological systems and for designing future therapeutics targeting iM DNA in cancer and related diseases.

The dysfunction of nucleocytoplasmic transport (NCT) and tau aggregation are emerging as interconnected hallmarks of neurodegenerative tauopathies. However, the molecular basis by which components of the nuclear pore complex and tau interact remains unclear. Here, we combine experimental and computational approaches to elucidate the mechanism of heterotypic phase separation between the FG-repeat domain of nucleoporin Nup98 (FG-Nup98) and tau. In vitro assays revealed that FG-Nup98 and tau undergo coacervation, forming dynamic condensates whose morphology and dynamics depend on stoichiometry, macromolecular crowding, and ionic strength. FRAP indicated reduced tau mobility within FG-Nup98-rich condensates, supporting a scaffold-client model. Complementary computational analyses revealed hierarchical binding energetics: FG-Nup98 self-association is strongest, followed by FG-Nup98-tau and tau-tau interactions. While FG-Nup98 forms stable homotypic networks, tau-tau contacts are transient but energetically favorable, which suggests that elevated tau concentrations may trigger a transition from droplets to tau aggregates. Together, these results establish that multivalent FG-Nup98-tau interactions drive condensate formation that could potentially perturb the permeability barrier of the nuclear pore. This study elucidates the coordinated behaviors of FG-Nup98-tau condensates and provides a framework for understanding NCT defects in tauopathies.

The inhibition of A{beta}42 ({beta}-amyloid) fibril formation is a key therapeutic strategy in Alzheimer's disease research. Paddlewheel diruthenium complexes have shown promising activity against A{beta}42 aggregation and preformed fibril disaggregation, yet their molecular mode of action remains poorly understood. In this work, we perform atomistic simulations to explore how charge modulation influences the interactions of three analogous paddlewheel diruthenium complexes, the parent neutral complex [Ru2Cl(D-p-FPhF)(O2CCH3)3], and its anionic [Ru2Cl2(D-p-FPhF)(O2CCH3)3]- and cationic [Ru2(D-p-FPhF)(O2CCH3)3]+ counterparts (D-p-FPhF- is the N,N' -bis(4-fluorophenyl)formamidinato ligand) with A{beta}42. Our results indicate that electrostatic tuning governs binding affinity and the extent of interaction across the A{beta}42 fibril surface. As the complexes' charge changes from -1 to +1, the interaction pattern shifts from localized contacts to widespread, multi-site engagement encompassing key charged, aromatic, and hydrophobic regions of A{beta}42. This enhanced binding correlates with longer-lived, thermodynamically stable interactions at the fibril interface, which effectively lower the free energy penalty for fibril disassembly. Overall, our findings propose a mechanism in which charge-dependent activation through ligand exchange enhances fibril recognition and promotes disruptive binding modes, demonstrating the potential of charge-tunable diruthenium complexes as therapeutic modulators of A{beta}42 fibril stability.

Apolipoprotein E (APOE) and Triggering Receptor Expressed on Myeloid cells 2 (TREM2) are the two strongest genetic risk factors of late-onset Alzheimers disease. ApoE binds to the low-density lipoprotein receptor (LDLR) to facilitate the uptake of ApoE-lipoprotein particles. TREM2 is a cell surface receptor expressed on microglia in the brain. The activation of TREM2 is essential for microglia to carry out protective functions against AD pathology. Several studies have shown that TREM2 signaling is activated through direct interaction between TREM2 and ApoE. In addition to its important role in AD pathogenesis, the ApoE/TREM2 interaction has been shown to induce immunosuppression of neutrophils within the tumor microenvironment. Despite its clinical importance, a high-resolution molecular understanding of the complex remains elusive. Here, we carried out chemical cross-linking mass spectrometry (XL-MS) analysis of the ApoE3/TREM2ECD complex to identify intra- and inter-protein cross-links, which were used as restraints to guide integrative protein-protein docking. Our data support a binding model in which a helix-loop-helix motif within the ApoE3 hinge and C-terminal region forms a transient hydrophobic pocket that wraps around the hydrophobic tip of the TREM2 ectodomain. This model is further supported by de novo-designed mini-protein binders, which show the same binding mode as identified by our XL-MS experiment. These results establish a robust framework for developing mini-protein-based TREM2 agonists.

Proteins often harness extensive motions of domains and subunits to promote their function. Deciphering how these movements impact activity is key for understanding lifes molecular machinery. The enzyme adenylate kinase is an intriguing example for this relationship; it ensures efficient catalysis by large- scale domain motions that lead to the enclosure of the bound substrates ATP and AMP. At high concentrations, AMP also operates as an allosteric inhibitor of the protein. Surprisingly, the enzyme is activated by urea, a compound commonly acting as a denaturant. Combining single-molecule FRET spectroscopy and enzymatic activity studies, we find that urea interferes with two key mechanisms that contribute to enzyme efficacy. First, urea promotes the open conformation of the enzyme, aiding the proper positioning of the substrates. Second, urea decreases AMP affinity, paradoxically facilitating a more efficient progression towards the catalytically active complex. These results signify the important interplay between conformational dynamics and chemical steps, including binding, in the activity of enzymes. State-of-the-art tools, such as single-molecule fluorescence spectroscopy, offer new insights into how enzymes balance different conformations to regulate activity.

Many enzymes display non-Arrhenius behaviour with curved Arrhenius plots in the absence of denaturation. There has been significant debate about the origin of this behaviour and recently the role of the activation heat capacity [Formula] has been widely discussed. If enzyme-catalysed reactions occur with appreciable negative values of [Formula] (arising from narrowing of the conformational space along the reaction coordinate), then curved Arrhenius plots are a consequence. To investigate these phenomena in detail, we have collected high precision temperature-rate data over a wide temperature interval for a model glycosidase enzyme MalL, and a series of mutants that change the temperature-dependence of the enzyme-catalysed rate. We use these data to test a range of models including macromolecular rate theory (MMRT) and an equilibrium model. In addition, we have performed extensive molecular dynamics (MD) simulations to characterise the conformational landscape traversed by MalL in the enzyme-substrate complex and an enzyme-transition state complex. We have crystallised the enzyme in a transition state-like conformation in the absence of a ligand and determined an X-ray crystal structure at very high resolution (1.10 [A]). We show (using simulation) that this enzyme-transition state conformation has a more restricted conformational landscape than the wildtype enzyme. We coin the term "transition state-like conformation (TLC)" to apply to this state of the enzyme. Together, these results imply a cooperative conformational transition between an enzyme-substrate conformation (ES) and a transition-state-like conformation (TLC) that precedes the chemical step. We present a two-state model as an extension of MMRT (MMRT-2S) that describes the data along with a convenient approximation with linear temperature dependence of the activation heat capacity (MMRT-1L) that can be used where fewer data points are available. Our model rationalises disparate behaviour seen for MalL and a thermophilic alcohol dehydrogenase and is consistent with a raft of data for other enzymes. Our model can be used to characterise the conformational changes required for enzyme catalysis and provides insights into the role of cooperative conformational changes in transition state stabilisation that are accompanied by changes in heat capacity for the system along the reaction coordinate. TLCs are likely to be of wide importance in understanding the temperature dependence of enzyme activity, and other aspects of enzyme catalysis.

The FET family proteins, which includes FUS, EWS, and TAF15, are RNA chaperones instrumental in processes such as mRNA maturation, transcriptional regulation, and the DNA damage response. These proteins have clinical significance: chromosomal rearrangements in FET proteins are implicated in Ewing family tumors and related sarcomas. Furthermore, point mutations in FUS and TAF15 are associated with neurodegenerative conditions like amyotrophic lateral sclerosis and frontotemporal lobar dementia. The fusion protein EWS::FLI1, the causative mutation of Ewing sarcoma, arises from a genomic translocation that fuses the low-complexity domain (LCD) of EWS (EWSLCD) with the DNA binding domain of the ETS transcription factor FLI1. This fusion not only alters transcriptional programs but also hinders native EWS functions like splicing. However, the precise function of the intrinsically disordered EWSLCD is still a topic of active investigation. Due to its flexible nature, EWSLCD can form transient interactions with itself and other biomolecules, leading to the formation of biomolecular condensates through phase separation - a mechanism thought to be central to the oncogenicity of EWS::FLI1. In our study, we used paramagnetic relaxation enhancement NMR, analytical ultracentrifugation, light microscopy, and all-atom molecular dynamics (MD) simulations to better understand the self-association and phase separation tendencies of EWSLCD. Our aim was to elucidate the molecular events that underpin EWSLCD-mediated biomolecular condensation. Our NMR data suggest tyrosine residues primarily drive the interactions vital for EWSLCD phase separation. Moreover, a higher density and proximity of tyrosine residues amplify the likelihood of condensate formation. Atomistic MD simulations and hydrodynamic experiments revealed that the tyrosine-rich N and C-termini tend to populate compact conformations, establishing unique contact networks, that are connected by a predominantly extended, tyrosine-depleted, linker region. MD simulations provide critical input on the relationship between contacts formed within a single molecule (intramolecular) and inside the condensed phase (intermolecular), and changes in protein conformations upon condensation. These results offer deeper insights into the condensate-forming abilities of the FET proteins and highlights unique structural and functional nuances between EWS and its counterparts, FUS and TAF15.

Fumarate-adding enzymes (FAE) are a subset of the glycyl radical enzyme superfamily involved in anaerobic hydrocarbon degradation. Benzylsuccinate synthase (BSS) catalyzes the enantiospecific formation of R-benzylsuccinate from toluene and fumarate, initiating anaerobic toluene degradation. In this paper, we present a detailed theoretical study of the reaction mechanism using classical molecular dynamics and multiscale modelling (QM:MM). We describe the potential energy surface of the reaction, confirming the previously postulated mechanism. However, the multiscale character of our model allowed to elucidate the origins of several experimentally observed catalytic phenomena, such as the inversion of the configuration of the benzylic atom upon C-C bond formation, syn addition of the abstracted H atom back to the benzylsuccinyl radical, or kinetic isotope effects in the range of 1.7-2.1. The obtained model is supported by microkinetic analysis and was able to explain and quantitatively predict the strict R-enantioselectivity of BSS, which is not enforced by the binding orientation of the fumarate, but by dynamic kinetic behaviour of toluene in the active site leading to faster production of the R-enantiomer. We were also able to explain the experimentally observed slow H/D exchange in the product during incubation with BSS in D2O, confirming the partial reversibility of the reaction. Our study contributes to the elucidation of the catalytic processes catalyzed by BSS and its role in the bioremediation of hydrocarbon pollutants.

Enzyme engineering has produced numerous methods to optimize enzymes for biotechnological processes; however, less is known about how natural evolution creates new functionalities. We investigate the evolutionary emergence of enantioselectivity in plant borneol dehydrogenases (BDHs), which feature hydrophobic active-sites and are enantioselective towards dibornane-type monoterpenols. Ancestral sequence reconstruction provided a trajectory from the oldest unselective BDH ancestor N30 (E=12) toward the youngest selective ancestor N32, involving 19 mutations: 18 mutations are peripheral, one (I111L) occurs in the active-site. The mutation L111I in the hydrophobic pocket increased the selectivity of N30, while the back-mutation I111L decreased the selectivity of N32. Additional peripheral mutations (V136L/G169A/V183I) were required for high selectivity. Crystal structures suggested that protein dynamics, rather than structural changes shape these catalytic properties. Molecular simulations with funnel-metadynamics revealed a correlation between the active-sites solvent-accessible surface area (SASA) and selectivity. This potential evolutionary pathway shapes enantioselectivity, and guides future enzyme engineering campaigns.

The propensity for intrinsically disordered proteins to aggregate is heavily influenced by their surrounding environment. Here, we show that the mobility of the surrounding water molecules directly influences the aggregation rate of -synuclein (aSyn), a protein associated with Parkinsons disease. We observe that the addition of NaCl reduces the mobility of water, while addition of CsI increases the mobility of water. In turn, this reduces and increases the mobility of aSyn, respectively, given the change in strength and lifetime of the intermolecular forces. The reduction of aSyn mobility in the presence of NaCl ions leads to increased aggregation rates, which may be due to aggregation-competent conformations being stable for longer, thereby increasing the likelihood of establishing interactions between two adjacent monomers. In contrast, aSyn is more mobile when CsI is dissolved in the aqueous phase which leads to a reduction of successful monomeric interactions. We thus highlight the importance of the surrounding environment and describe how ion content can influence water mobility and the misfolding rate of amyloidogenic proteins, such as aSyn. By modulating the cellular environment to increase water mobility or finding small molecules to increase protein dynamics, new therapeutic targets may be found.

The eukaryotic positive transcription elongation factor b (P-TEFb), composed of CDK9 and cyclin T, plays a central role in regulating RNA polymerase II (RNAPII). Phosphorylation of the RNAPII C-terminal domain (CTD) by P-TEFb promotes promoter proximal pause release and enables productive transcriptional elongation across many genes. Cyclin T mediates protein-protein interactions, several of which have been structurally characterised, that help to recruit and fine-tune P-TEFb activity to ensure a tight regulation of transcription. We have previously reported a set of halogenated chemical fragments termed FragLites that can prospectively identify protein interaction sites. Here, we report the FragLite map of cyclin T2, revealing binding sites corresponding to structurally defined cyclin T partners CDK9, AFF4, and HIV-1 Tat. Furthermore, we demonstrate the utility of FragLites in identifying a previously uncharacterised BRD4 binding site. By integrating FragLite clustering with biophysical analyses and AlphaFold3 modelling, we delineate the cyclin T-BRD4 interface. These analyses provide a comprehensive, chemically enriched fragment map highlighting functionally relevant sites to support future probe and modulator development to selectively target P-TEFb.

Fluorescent proteins (FP) are frequently used for studying proteins inside cells. In advanced fluorescence microscopy, FPs can report on additional intracellular variables. One variable is the local density near FPs, which can be useful in studying densities within cellular bio-condensates. Here, we show that a reduction in fluorescence lifetimes of common monomeric FPs reports increased levels of local densities. We demonstrate the use of this fluorescence-based variable to report the distribution of local densities within heterochromatin protein 1 (HP1) in mouse embryonic stem cells (ESCs), before and after early differentiation. We find that local densities within HP1 condensates in pluripotent ESCs are heterogeneous and cannot be explained by a single liquid phase. Early differentiation, however, induces a change towards a more homogeneous distribution of local densities, which can be explained as a liquid-like phase. In conclusion, we provide a fluorescence-based method to report increased local densities and apply it to distinguish between homogeneous and heterogeneous local densities within bio-condensates.