JACS Au

Thermal proteome profiling (TPP) and its higher-throughput derivative, the proteome integral solubility alteration (PISA) assay, measure changes in protein thermal stability upon ligand binding or other perturbations and have been widely adopted in drug discovery and biomedical research. Though the PISA workflow is straightforward, key parameters, including detergent concentration, methods for removing denatured aggregates, and temperature range selection, vary across studies and can markedly influence assay outcomes. Yet these factors have not been systematically evaluated, limiting rational experimental design and data interpretation. Here, through a combined use of TPP, PISA, tandem mass tag (TMT)-based multiplexing, and computational simulation, we systematically characterize these parameters based on the melting behavior of [~]9,000 proteins. We find that reducing detergent concentration elevates apparent Tm by 1.5-2{degrees}C proteome-wide, and aggregate removal by filtration versus centrifugation further alters measurements. We leverage these observations to optimize PISA then apply the optimized conditions to identify the aminopeptidase NPEPPS as a previously uncharacterized binding partner of angiotensin II, a key vasoactive peptide hormone in blood pressure regulation. Together, this work provides a general framework for assay design and data interpretation, and extends the utility of PISA beyond small molecules to dissecting peptide-protein interactions, an increasingly important modality in drug discovery.

Solid-phase peptide synthesis (SPPS) remains the dominant technique for peptide production. However, its reliance on hazardous organic solvents such as N, N-dimethylformamide (DMF) and dichloromethane (DCM) results in an adverse environmental burden. One potential approach is replacing these organic solvents with water to reduce the hazardous solvent consumption and improve the environmental footprint of peptide production. This has led to the emergence of aqueous solid-phase peptide synthesis (ASPPS) approaches. Although successful, these approaches require specialized hydrophilic resins or modified building blocks, limiting their industrial applicability and scalability. Moreover, conventional hydrophobic polystyrene supports, remain the most widely used solid supports in industrial SPPS due to their high loading capacity, mechanical robustness, and low cost. These resins are generally considered incompatible with aqueous conditions. Here, we demonstrate that industrially relevant 2-chlorotrityl chloride (CTC) polystyrene resin can support efficient peptide coupling under fully aqueous conditions by integrating a precipitate-free 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC{middle dot}HCl) and Oxyma activation system with a synergistic thermal-acoustic strategy. We posit that heating combined with ultrasonic irradiation likely promotes transient relaxation of the polystyrene matrix and enhances water penetration. This facilitates the diffusion of activated amino acid esters onto the hydrophobic resin required for coupling. The robustness of this aqueous methodology was validated through the synthesis of nine structurally diverse peptide sequences, including aromatic hydrogel-forming peptides, opioid peptides derived from enkephalins, toxin-inspired sequences, and a lipid-interacting fragment of -synuclein. Analytical characterization by HPLC and MALDI-TOF mass spectrometry confirmed successful peptide assembly with high crude purity. We anticipate that this thermal-acoustic aqueous SPPS strategy provides a scalable and accessible pathway toward sustainable peptide manufacturing on classical hydrophobic supports with aqueous chemistry.

O2-tolerance is a desirable property for [FeFe] hydrogenases, which are highly efficient H2-producing catalysts. While most such enzymes are highly sensitive to aerobic environments, a small number of explored representatives exhibit exceptional stability and even H2-producing activity under oxygenic conditions. However, the genetic signatures of the O2-tolerance in this class of enzymes remain largely unknown. To address this knowledge gap, we explored a close homologue of a well-characterized O2-tolerant [FeFe] hydrogenase from Clostridium beijerinckii (CbHydA1) - a hydrogenase from Terrisporobacter glycolicus (TgHydA1). Our investigation indeed confirms that TgHydA1 can transition to the O2-stable Hinact state, a hallmark of O2 tolerance. The surprising outcome is that despite the high amino acid similarity, TgHydA1 shows a substantially higher propensity to remain in the Hinact state than CbHydA1. Using protein film electrochemical experiments, we demonstrate that the root of this behavior lies in roughly tenfold slower reactivation rates than those of CbHydA1 at any applied potential. This degree and direction of variation in reactivation kinetics have not been observed before for any other O2-tolerant [FeFe] hydrogenases or their variants to date, uncovering a yet-to-be-explored facet of reactivity alteration available to these enzymes. Overall, the results presented here highlight the importance of a holistic analysis of [FeFe] hydrogenase sequences in the context of their interaction with O2 that encompasses the protein environment and properties of the auxiliary metallocofactors.

Detergent-free extraction of membrane proteins using polymers directly into nanodiscs from the cell membrane has been used widely in recent years. Since the first use of poly(styrene-co-maleic acid) (SMA), numerous related polymers have been developed that differ in chemical architecture and nanodisc characteristics, each capable of influencing the structural and functional properties of the encapsulated membrane protein and its surrounding lipids. Identifying an optimal solubilising polymer, therefore, requires consideration not only of extraction efficiency but also compatibility with downstream applications and analyses. Polymer series in which a single parameter is systematically varied provide a valuable, nuanced tool for optimising nanodisc utility in downstream applications. This study utilises a chemically defined series of poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers that exhibit a stepwise, systematic increase in hydrophobicity. Using the human calcitonin gene-related peptide (CGRP) receptor as an exemplar class B1 G-protein-coupled receptor (GPCR), the ability of each BzAM terpolymer to solubilise the receptor from mammalian cell membranes was assessed. All members of the series successfully solubilised CGRP receptor, with solubilisation efficiency correlating positively with increasing hydrophobicity. Importantly, the receptor retained its characteristic high-affinity ligand-binding capability when encapsulated within the BzAM nanodisc, demonstrating that functional integrity is preserved following BzAM-mediated extraction and purification. These findings establish the BzAM terpolymer series as a systematic, tuneable, well-defined tool for the detergent-free solubilisation and functional investigation of GPCRs, and other membrane proteins, in near-native lipid environments. HIGHLIGHTSO_LIStepwise-tuned poly(styrene-co-maleic acid-co-(N-benzyl)maleimide) (BzAM) terpolymers provide a chemically defined, hydrophobicity-controlled platform for detergent-free membrane protein extraction. C_LIO_LIAll BzAM variants effectively solubilise the human calcitonin gene-related peptide (CGRP) receptor, with extraction efficiency increasing in line with terpolymer hydrophobicity. C_LIO_LICGRP receptor maintains high-affinity ligand binding in BzAM nanodiscs, demonstrating preservation of ligand-binding function after solubilisation. C_LIO_LIThe BzAM series provides a novel platform for studying G-protein-coupled receptors and other membrane proteins in near-native lipid environments, with the potential to deliver mechanistic insights and support future drug-discovery efforts. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/726474v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1cb167corg.highwire.dtl.DTLVardef@313e60org.highwire.dtl.DTLVardef@f64a2borg.highwire.dtl.DTLVardef@17f6629_HPS_FORMAT_FIGEXP M_FIG C_FIG

In todays world, point-of-care nucleic acid detection still remains extensively constrained and limited by the heavy dependence on centralized urban instrumentation facilities and complex assay workflows. Here, we elucidate a glucometer-based analytical platform that enables label-free detection of nucleic acids and the nucleic acid amplification products through a simple redox-mediated mechanism. The approach leverages the potassium ferricyanide (K3[Fe(CN)6])/ potassium ferrocyanide (K4[Fe(CN)6]), redox system, which is intrinsic to commercial glucometers, complementing with interactions between methylene blue (MB) and nucleic acids. These interactions transduce concentration differences in nucleic acids into quantifiable electrochemical signal readouts. Distinct varied signal outputs are observed between single-stranded and double-stranded DNA, enabling the direct detection as well as integration with nucleic acid amplification tests (NAATs), including polymerase chain reaction, rolling circle amplification, and loop-mediated isothermal amplification. Optimization of reaction parameters and conditions leads to enhancement of the overall signal discrimination and sensitivity across various assay formats. This innovation repurposes widely available off-the-shelf glucometers as a low-cost, portable nucleic acid detectors, thus eliminating the need for any specialized instrumentation. Our results enumerate and establish a generalized and scalable strategy for nucleic acid sensing. The platform thus supports sustainable and environmentally responsible point-of-care testing, thereby enabling improved accessibility and public health monitoring at resource-limited and remote settings.

The nuclear receptor superfamily is comprised of ligand-regulated transcription factors that contain an intrinsically disordered domain at the amino-terminal end, known as the N-terminal domain (NTD). While this poorly conserved domain is known to possess ligand-independent activation function (AF-1), few NTD functions are conserved between nuclear receptors (NRs). Identified roles in other receptors include androgen receptor (AR), estrogen receptor (ER) and mineralocorticoid receptor (MR). Here, we aim to define the function of the NTD of the farnesoid X receptor (FXR), a crucial regulator of lipid and bile acid metabolism. We show that the NTD engages in interdomain contact with other FXR domains. We also observe that the NTD interacts directly with coregulator proteins. Using mutagenesis, mammalian two-hybrid assays and molecular dynamics simulations, we identify and validate a novel SXXLF motif in the NTD which mediates interactions with both coregulators and the ligand binding domain. Mutation of the motif induces large changes in conformational and allosteric coupling in FXR. Our study identifies a new nuclear receptor-interacting motif that modulates the transcriptional activity of FXR. Graphical AbstractFXR-NTD regulates transcriptional activity through interdomain communication with the LBD and is also involved in co-activator recruitment. The SENLF motif is the first defined functional element within the FXR-NTD and mediates both NTD-LBD interaction and selective co-activator engagements to drive NTD-mediated transcriptional activity. O_FIG O_LINKSMALLFIG WIDTH=135 HEIGHT=200 SRC="FIGDIR/small/724725v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@5a37aorg.highwire.dtl.DTLVardef@2fa9e1org.highwire.dtl.DTLVardef@13a19daorg.highwire.dtl.DTLVardef@1775ed2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Native mass spectrometry (nMS) is a powerful tool for analyzing biomolecules and their complexes under near native conditions. The preservation of the native state depends strongly on the ionization methods used to transfer intact molecules from solution to gas phase. In this work, capillary vibrating sharp-edge spray ionization (cVSSI)- based nMS and in-droplet hydrogen deuterium exchange mass spectrometry (HDX-MS) were used to evaluate calcium-dependent interactions between calmodulin and calmidazolium (CDZ). We found that cVSSI produced a narrow charge-state-distribution (CSD) with low average charge states indicating that this method preserved the native-like state. cVSSI was also able to resolve stepwise Ca2+-binding containing one to four Ca2+-bound species of the protein. In absence of Ca2+, no detectable CDZ-binding was observed. However, CDZ-binding was observed when calmodulin was fully loaded with Ca2+. CDZ-binding to the protein caused marked redistribution of the CSD toward lower charge states, consistent with ligand-induced stabilization of the protein into a more compact conformation. The apparent dissociation constant (Kd) of the interaction was determined to be 261 {+/-} 29 nM and 126 {+/-} 17 nM from Langmuir and quadratic binding models, respectively. Complementary in-droplet HDX-MS showed an approximately 23% reduction in deuterium uptake upon ligand binding indicating reduced solvent accessibility and increased structural stabilization supporting nMS findings. Together, these results demonstrate that cVSSI-based nMS coupled with in-droplet HDX-MS provides an integrated platform for simultaneously resolving metal loading, ligand binding, binding affinity, and ligand-induced conformational changes. This approach complements traditional structural methods by enabling direct interrogation of dynamic, metal-dependent protein-ligand interactions in their native states.

Deep characterization of intact proteoforms remains an analytical challenge in functional proteomics, particularly for heterogenous multi-site post-translational modifications at distinct amino acid residues. Histones are among the most dynamically and diversely post-translationally modified proteins in eukaryote cells, carrying multiple, co-occurring and reversible modifications that can give rise to isomeric proteoform species. Tandem mass spectrometry with multimodal fragmentation capabilities is a promising approach for deep characterization of intact proteoforms, such as modified histones. We applied the novel timsOmni mass spectrometer, which incorporates the Omnitrap platform enabling multimodal MS workflows, for residue-level mapping of histone modifications, including acetylation and methylation. Recombinant histones H3.1 and H4 were in vitro acetylated by enzymes GCN5, PCAF and p300 to generate mono- and multi-acetylated proteoforms. Complementary MS2 electron- and collision-based dissociation (ECD, EID, RCID and ECciD), together with MS3 strategies, produced complete or near-complete backbone fragmentation of intact protein ions (>92% amino acid sequence coverage). For monoacetylated species generated by the more site-selective lysine acetyltransferases, the dominant proteoform matched the known catalytic preferences of the enzymes (H3.1K14ac for GCN5 and PCAF, and H4K8ac for PCAF), while minor positional isomers were also identified and their relative abundance estimated. In contrast, the broader substrate specificity of p300 produced a wide distribution of H4 proteoforms bearing up to seven acetylated lysine residues. Species carrying six and seven acetylations were characterized by multimodal MS2/MS3 experiments, enabling localization of individual acetylation sites and discrimination of positional isomers. Finally, endogenous histone proteoforms from liver extracts were analyzed, yielding sequence coverages of 92-93% for the most abundant species and enabling confident localization of multiple PTMs (acetylation and methylation). These results illustrate that multimodal MSn fragmentation of intact proteins supports residue-level assignment of combinatorial histone marks and coexisting positional isomers. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=165 HEIGHT=200 SRC="FIGDIR/small/722147v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@387ab5org.highwire.dtl.DTLVardef@2410org.highwire.dtl.DTLVardef@13fc392org.highwire.dtl.DTLVardef@140e054_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMultimodal MS{superscript 2}/MS3 maps histone PTMs on intact proteins. C_LIO_LIECD, EID, RCID, and ECciD provide complete or near-complete sequence coverage. C_LIO_LIMS3 localizes acetylation sites, distinguishes positional isomers. C_LIO_LIEndogenous H4 proteoforms are assigned with site-specific PTM mapping. C_LI

DNA topology is a key regulator of chromatin structure and transcription, yet its direct role in transcription factor recognition remains unclear. Here, we investigate how distinct DNA topological states modulate binding of the Saccharomyces cerevisiae bZIP transcription factor GCN4 using topologically defined plasmids. By combining, complementary biochemical approaches, including Bio-Layer Interferometry applied here for the first time to topology-dependent protein-DNA interactions, we show that DNA supercoiling directly reshapes GCN4-DNA recognition. Positively supercoiled DNA forms more stable and persistent complexes, whereas negatively supercoiled DNA retains greater conformational heterogeneity. To interpret these effects, we performed multiscale molecular simulations. Coarse-grained simulations of plasmids recapitulate the global topology-dependent trends observed experimentally, while matched minicircle models reproduce the same behaviour at the local scale. In strong agreement with experimental data, simulations reveal that DNA topology modulates the conformational ensemble of the GCN4 basic region. Overall, positively supercoiled DNA promotes a more ordered binding mode and localized protein distribution, whereas negatively supercoiled DNA supports increased structural plasticity. These findings identify DNA topology as an active determinant of transcription factor recognition and provide a multiscale framework linking global DNA mechanics to local protein-DNA interactions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/722604v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@18f8ba9org.highwire.dtl.DTLVardef@11a395dorg.highwire.dtl.DTLVardef@ac093borg.highwire.dtl.DTLVardef@923212_HPS_FORMAT_FIGEXP M_FIG C_FIG

c-Myc is a transcription factor that drives tumorigenesis in many cancers. It is notoriously difficult to directly target c-Myc, mainly due to its lack of well-defined druggable pockets. O-linked {beta}-N-acetylglucosamine modification (O-GlcNAcylation) is a post-translational modification (PTM) playing an important role in regulating c-Myc functions in cancer. However, previous studies have primarily relied on global perturbations to investigate c-Myc O-GlcNAcylation, making it difficult to determine its direct functional consequences due to concurrent cellular effects. Here, we report a bifunctional O-GlcNAcylation TArgeting Chimera (OGTAC) molecule, which can induce the proximity of c-Myc and O-GlcNAc transferase (OGT) in living cells, thereby enhancing the O-GlcNAcylation of c-Myc. The c-Myc-targeting OGTAC exhibits anti-proliferation effect against cancer cells. Mapping of c-Myc occupancy on genome indicates that OGTAC rewires c-Myc transcriptional activity and reprograms expression of the downstream oncogene MALAT1, in an O-GlcNAcylation-dependent manner. Overall, OGTAC presents a novel chemically induced proximity (CIP)-based tool to target and rewire c-Myc activity in cancer. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/722559v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@d1c640org.highwire.dtl.DTLVardef@2eb70corg.highwire.dtl.DTLVardef@f38970org.highwire.dtl.DTLVardef@c421c8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Conformationally responsive DNA nanoswitches have previously been developed and validated for a variety of biosensing applications including detection of DNA, microRNA, and viral RNA/DNA. Here we develop new methodology for enhancing the sensitivity of DNA-based sensing by recycling a fixed number of targets for repeated reuse. We achieved target-dependent enzymatic ligation of looped nanoswitches and showed that subsequent removal of target does not affect the ligated loop. Through cyclic annealing, ligation, and target removal, we can linearly control signal amplification up to hundreds of cycles. This method adds an important new capability for low abundance targets without the need for target amplification.

Transfer RNA methyltransferase 1 (TRMT1) installs N2-methylguanosine and N2,N2-dimethylguanosine modifications at position 26 of mammalian tRNAs, supporting tRNA structure, translation, and cellular response to redox stress. However, the local environment and interactome of TRMT1 in the cell is poorly defined. Here, we use APEX2-based proximity labeling of the N- and C-terminus of TRMT1, coupled with label-free quantitative proteomics to map candidate TRMT1-proximal proteins in HEK293T cells. Mass spectrometry data was acquired using both data-independent acquisition (DIA) and data-dependent acquisition (DDA) methods, and it was found that DIA substantially increased proximity proteome coverage, reproducibility, and the number of significantly enriched candidate hits compared to the DDA method. N- and C-terminal APEX2-TRMT1 constructs captured largely overlapping proteomes, suggesting the dual-labeling strategy provides a robust map of proximal proteins. Analysis of the significant TRMT1-proximal proteins reveals enrichment in RNA processing and ribonucleoprotein-associated factors, in addition to hits connected to tRNA modification, tRNA biogenesis, and redox-associated biology. These data provide a proteome-scale view of TRMT1-associated cellular proteins and environments, and lay the groundwork for future validation of functional TRMT1 interaction networks. SignificanceO_LIFusing APEX2 enzyme to both N-terminal and C-terminal of the bait enhanced the sensitivity for identification of protein interactions. C_LIO_LICombining APEX2-based endogenous labeling with DIA mass spectrometry increases reproducibility and depth of proximity proteome. C_LIO_LIThe study provides a rich source of potential interacting or proximally close proteins to TRMT1, which warrants further validation studies. C_LI

The Apolipoprotein E4 (ApoE4) genotype is the most significant genetic risk factor for late-onset Alzheimers disease (AD). A key driver of ApoE4 cellular toxicity is the endo-lysosomal burden resulting from the excessive receptor-mediated uptake of ApoE4 lipoparticles. The high-affinity interaction between lipidated ApoE4 and the Low-Density Lipoprotein Receptor (LDLR) saturates the cellular degradation machinery, correlating with lysosomal alkalinization, lipid accumulation, and cell death. To target this critical interaction interface, which consists of 7 tandem ligand-binding type-A (LA) modules in the human LDLR, we present the design and evaluation of recombinant LDLR minireceptors comprising combinations of these LA modules to competitively antagonize ApoE4 endocytosis. We observe a distinct isoform-dependent uptake dynamic across multiple central nervous system (CNS) cell models, with ApoE4 showing significantly greater total intracellular accumulation than ApoE2. Furthermore, engineered LA peptides selectively bind ApoE4 over human serum LDL and differentially inhibit its uptake, revealing a distinct structural efficacy hierarchy of LA3456 [~] LA345 > LA456 [~] LA45 >> LA34. We establish the resilience of the LA45 minireceptor under physiological serum conditions and identify LA345 as the most stable truncated construct in vitro. Notably, molecular tagging orientation is critical for therapeutic engineering; C-terminal tagging completely preserves the inhibitory function of the minireceptors, whereas N-terminal tagging drastically reduces it. These findings provide a framework for scalable, deliverable inhibition of the ApoE4-LDLR interaction as a potential therapeutic target to mitigate endo-lysosomal accumulation in AD.

Peptide mapping is a critical tool for characterizing biotherapeutic proteins and is essential for the development of monoclonal antibody drugs. Here we describe a new direct infusion technology that streamlines peptide mapping data collection and analysis, accelerating the method by up to 100-fold. This method, which we term RaPiD-mAb-MS, combines high-throughput plate-based sample preparation with direct infusion mass spectrometry analysis. RaPiD-mAb-MS allows analysis of 96 samples within [~] 1.5 to 2 hours, routinely achieves >95% sequence coverage, and has been successfully applied to 28 unique antibodies and over 2,000 samples. Here we demonstrate that RaPiD-mAb-MS detects and quantifies oxidation, deamidation, isomerization, glycosylation, and sequence variants with results comparable to conventional LC-MS based methods in a fraction of the time. Further, by eliminating chromatography, data analysis is greatly streamlined and simplified. By allowing for the collection of [~] 1,000 peptide maps per day, RaPiD-mAb-MS is positioned to accelerate all phases of antibody-based drug discovery & development and sets the stage for collection of massive datasets that would allow artificial intelligent prediction of optimal antibody variants and formulations.

Antibiotic resistance in Mycobacterium tuberculosis is a pressing global health challenge demanding new therapeutic strategies. The bacterial respiratory chain comprises promising antibacterial targets, with dual inhibition of the terminal oxidases cytochrome bcc:aa3 and cytochrome bd (cyt bd) showing bactericidal activity. While bcc:aa3 inhibitors such as Q203 have advanced clinically, cyt bd remains underexplored due to difficulties in assigning activity of the purified enzyme and structurally resolving the quinol substrate binding site. Here, we report a rapid in vitro screening platform for cyt bd inhibitors by engineering a minimal respiratory system that couples the activity of cyt bd to that of a type 2 NADH dehydrogenase. This coupled assay enables spectroscopic monitoring of NADH oxidation as a proxy for cyt bd activity, allowing rapid screening of over 10,000 compounds. Screening identified WSL017, a fragment with low micromolar potency against both M. tuberculosis and E. coli cyt bd. Kinetic and structural analyses revealed competitive inhibition at the quinol-binding site, providing the first structural insights into cyt bd inhibition by a non-quinone scaffold. WSL017 displayed growth inhibition of M. tuberculosis H37ra, corroborating oxidase inhibition as a promising therapeutic strategy. This work establishes a pipeline for cyt bd inhibitor discovery and highlights new opportunities for structure-guided drug development against cytochrome bd oxidases.

Understanding proteoforms, i.e., the various molecular forms in which proteins can exist, is important for deciphering biological processes and diseases. While capillary zone electrophoresis (CZE) proved advantageous for proteoform separation, limited sample loading capabilities restrict its application. Here, we present a novel comprehensive two-dimensional nanoLCxCZE-MS platform for deep top-down proteomics (TDP). The 2D platform is highly automated, enabling robust performance and the possibility to perform proteoform quantitation as demonstrated by isobaric labeling experiments. The high orthogonality of reversed-phase LC and CZE leads to a peak capacity of 2200, leading to an increase in the number of identified proteoforms in a human Caucasian colon adenocarcinoma cell lysate sample by a factor of 3 compared to nanoLC-MS. Furthermore, CZE mobilities enable the attribution of many more proteoforms to a certain proteoform family on the MS1-level. Overall, the flexible platform enables highly efficient separation of intact proteoforms combined with sensitive MS-based TDP workflows, both for untargeted and targeted analysis of complex biological samples. Graphical AbstractWe report a robust and automated comprehensive nanoLCxCZE-MS platform for top-down proteomics. In addition to large volume sample injection and separation by hydrophobicity in the nanoLC, the orthogonal separation by CZE in the second dimension leads to a strong increase in peak capacity and, thus, in the number of identified proteoforms. CZE mobilities also enable the attribution of many more proteoforms to a proteoform family on the MS1-level. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=46 SRC="FIGDIR/small/725123v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@df07b6org.highwire.dtl.DTLVardef@736d5corg.highwire.dtl.DTLVardef@10cef1org.highwire.dtl.DTLVardef@1825b55_HPS_FORMAT_FIGEXP M_FIG C_FIG

Protein tyrosine kinases are important regulators of cell signaling, and aberrant kinase activity contributes to many human diseases, including cancers. All protein tyrosine kinases share a highly-conserved ATP binding pocket but diverge in their substrate binding sites in order to mediate distinct signaling events. Many potent and efficacious ATP-competitive tyrosine kinase inhibitors have been developed, however it remains challenging to achieve on-target selectivity across different kinases and target specific disease mutants, given the high degree of conservation in the ATP-binding pocket. By contrast, the variable substrate-binding site offers an opportunity for selective inhibition, provided molecules can be targeted to this site. Here, we present a modular strategy to design selective, peptide-based covalent inhibitors of tyrosine kinases with a distinct binding mode from existing ATP-competitive inhibitors. Using Src kinase as a model system, we demonstrate that Src-selective reactivity can be achieved by first designing an optimized substrate peptide and then strategically positioning an electrophile on the peptide to target a non-conserved cysteine on the kinase. We show that substrate-derived covalent peptides can inhibit kinase activity, bind simultaneously with an ATP-competitive inhibitor, and even inhibit the activity of kinases bearing a common drug resistance mutation. We further explore the application of this approach to develop an inhibitor of the cancer-relevant fibroblast growth factor receptor 1 kinase that shows selectivity for an oncogenic mutant over the wild-type enzyme. Our modular strategy to generate selective covalent peptides targeting protein tyrosine kinases provides a promising framework for future chemical probe and drug development efforts.

Biomolecular condensates formed by intrinsically disordered proteins (IDPs) rely on a balance of sequence-encoded interactions and secondary-structure elements. TDP-43, a disease-associated protein, undergoes liquid-liquid phase separation (LLPS) through its low-complexity domain, whereas Hero11 has been proposed to modulate its condensate properties. However, the molecular mechanisms by which Hero11 affects the internal organization and dynamics of TDP-43 condensates remain unknown. Here, using multi-microsecond explicit-solvent all-atom simulations spanning single chains to =~100-chain condensates, we show that the TDP-43 -helix, which is only marginally stable in isolation, becomes a major structural hub within the condensate, forming a percolated helix-helix interaction network whose contact lifetimes are substantially longer than those of the surrounding disordered contacts. Hero11 selectively dismantles this network: it binds preferentially near the helical region, reduces the helix-helix coordination number, and shortens helix-helix contact lifetimes. This targeted disruption lowers condensate density, increases both water and ion infiltration, and enhances TDP-43 diffusion within the dense phase. Notably, dimer simulations reveal that the interactions between TDP-43 and Hero11 are too weak to persist under dilute conditions, indicating that the regulatory effect emerges only through multivalent contacts in the condensed phase. These results establish the -helix as a selectively vulnerable structural element within the TDP-43 condensate and provide an atomic-level mechanism for how a highly charged disordered protein can tune condensate material properties by targeting its longest-lived interaction nodes.

Chlamydia trachomatis is an obligate intracellular Gram-negative pathogen responsible for sexually transmitted infections and trachoma in humans. Although antibiotics are generally effective against acute infections, persistent chlamydial forms often exhibit reduced susceptibility during chronic infection. Chlamydia relies on its type III secretion system (T3SS) to inject effector proteins into host cells, making T3SS proteins attractive targets for antivirulence therapeutics. In this study, we employed an integrated computational pipeline to model and assemble the C. trachomatis T3SS constituent proteins. Template-based modeling using crystallographic structures of homologs from other Gram-negative bacteria revealed a highly conserved structural architecture despite low sequence identity (18-46%). Stereochemical validation confirmed high model quality, with most T3SS proteins exhibiting favorable protein-protein interactions (PPIs). Since the activity of the T3SS complex relies on extensive PPIs, we targeted these PPIs as a promising approach to attenuate bacterial virulence. CdsN, which functions as an ATPase of the T3SS, is a hexamer of which we targeted the dimerization interface. Structure-based virtual screening of compounds from the e-Drug3D and IMPPAT libraries against predicted hotspot residues and the identified druggable pocket at the CdsN dimeric interface, followed by ADMET screening, yielded three promising candidates: M Roflumilast (Drug ID: 1537), Elacestrant (Drug ID: 2081), and Tecovirimat (Drug ID: 1889). All three ligands formed thermodynamically stable complexes with the CdsN dimer, with Elacestrant demonstrating the most favourable binding free energy. This was also confirmed by 100 ns molecular dynamics simulation. This study provides new insights into the molecular architecture of C. trachomatis T3SS and identifies M Roflumilast, Elacestrant, and Tecovirimat as potential drug candidates against chlamydial infection. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/723290v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@1821599org.highwire.dtl.DTLVardef@1581baaorg.highwire.dtl.DTLVardef@1805e98org.highwire.dtl.DTLVardef@c25e56_HPS_FORMAT_FIGEXP M_FIG C_FIG

Intrinsic tryptophan fluorescence is widely used as a sensitive reporter of protein conformational dynamics, yet the molecular origin of its temperature-dependent modulation remains unclear. Here we investigate the conformational dynamics of Trp134 in bovine serum albumin (BSA) using molecular dynamics (MD) simulations, free-energy calculations based on umbrella sampling and WHAM, quantum mechanical (QM) calculations, and QM/MM approaches. MD simulations show that the global structure of BSA remains stable while temperature induces a gradual population shift from the Ia+ to the Ia- rotamer. The corresponding free-energy landscapes reveal that this shift arises from subtle changes in basin stability and transition barriers along the rotameric coordinate. In contrast, standalone QM calculations on isolated tryptophan predict different energetic trends, highlighting the sensitivity of rotamer stability to electronic-structure treatments and environmental effects. QM/MM calculations partially reconcile these differences by incorporating the protein environment. Together, these results suggest that temperature reshapes the rotamer free-energy landscape of Trp134, leading to population shifts that modulate intrinsic tryptophan fluorescence in proteins.