JACS Au

Affinity purification is a essential technique for isolating highly purified proteins; however, generating affinity ligands require significant time and financial investment. To address these limitations, this study proposes a novel affinity chromatography method utilizing in silico-designed cyclic peptides as ligands. Targeting Complement C1q (C1q), a plasma protein that plays crucial roles in classical complement pathway, we employed the biomolecular structure prediction model, AlphaFold2, to design specific binding cyclic peptides. Based on these designs, we synthesized lariat-type cyclic peptides characterized by disulfide cyclization and biotinylation, which were subsequently immobilized on streptavidin carriers. Performance tests confirmed that the resulting column specifically captured C1q, allowing for elution via a standard NaCl concentration gradient. Notably, high selectivity was preserved even in the presence of plasma, underscoring the ligands practical robustness. By overcoming traditional constraints through (1) rapid and simple design, (2) high specificity, and (3) universal versatility without genetic modification, this de novo design strategy represents a potential breakthrough in protein purification technologies. HighlightsO_LIAI-driven de novo design generated a specific cyclic peptide ligand for Complement C1q C_LIO_LIThe synthetic ligand enabled one-step purification of Complement C1q directly from human plasma C_LIO_LIMild elution conditions preserved the targets oligomeric structure and native interactome C_LIO_LIThis label-free strategy offers a rapid, low-cost alternative to antibody-based chromatography C_LI

In this study, we demonstrate that urea enables reliable melting temperature (Tm) determination of hyperthermostable proteins by nano differential scanning fluorimetry (nanoDSF) Under native conditions, Pfu DNA polymerase and its Sso7d-fusion variant showed no detectable unfolding transitions, despite their Tm values falling within the instruments operational range, reflecting their extreme kinetic stability. In the presence of up to 7 M urea, intrinsic tyrosine and tryptophan fluorescence revealed clear unfolding transitions, yielding extrapolated Tm values of 104.8 {+/-} 0.09 {degrees}C for Pfu and 106.8 {+/-} 0.33 {degrees}C for its Sso7d-fusion variant. These results demonstrate that urea-gradient nanoDSF overcomes both instrumental and kinetic limitations, providing a simple and robust method for assessing the thermal stability of (hyper)thermostable proteins. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/717478v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@960d9org.highwire.dtl.DTLVardef@1b5613forg.highwire.dtl.DTLVardef@1039a08org.highwire.dtl.DTLVardef@1759841_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

GPR52 is an orphan G protein-coupled receptor implicated in psychiatric and neurodegenerative disorders, but its endogenous ligand remains unidentified, limiting the exploration of its physiological functions and therapeutic potential. We pioneered a novel methodology for orphan GPCR ligand discovery utilizing the GPCR-activation-based (GRAB) strategy by developing GPR52-1.0, a genetically encoded fluorescent sensor. GPR52-1.0 exhibits excellent membrane trafficking and high sensitivity in HEK293T cells, cultured neurons, and acute mouse brain slices. Notably, it detects neuronal activity-dependent endogenous ligand release in the striatum, with responses abolished by a specific antagonist. This sensor provides a powerful tool for identifying GPR52s endogenous ligand(s) and enables real-time monitoring of its activation. Our work lays the foundation for uncovering GPR52s physiological roles and supports future efforts to develop GPR52-targeted therapeutics.

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.

Per- and polyfluoroalkyl substances (PFAS) are highly resistant to enzymatic C-F bond cleavage, and hydrolytic defluorination of long-chain PFAS has rarely been demonstrated. Here, we report selective hydrolytic defluorination of branched perfluorooctanoic acid (PFOA) isomers by a haloacid dehalogenase (4A) from Delftia acidovorans strain D4B. A fluoride-specific riboswitch biosensor was used for initial substrate screening, followed by scaled-up assays in which fluoride release was quantified using a fluoride ion-selective electrode. Defluorination products were subsequently identified by liquid chromatography-mass spectrometry (LC-MS). Although purified 4A (10 M) readily catalyzed hydrolytic defluorination of fluoroacetic acid, incubation of PFOA (0.5 mM) with purified 4A resulted in a statistically significant increase in fluoride release at elevated enzyme loading (500 M). High-resolution LC-MS/MS analysis revealed that defluorination products originated from minor branched PFOA isomers rather than linear PFOA. Molecular docking analyses supported catalytically plausible binding geometries for branched PFOA isomers, positioning the substrate -carbon within [~]4 [A] of the catalytic aspartate residue. These findings demonstrate previously unrecognized hydrolytic reactivity of a haloacid dehalogenase toward branched PFAS isomers and expand the known catalytic scope of the haloacid dehalogenase family. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/719434v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@16da75dorg.highwire.dtl.DTLVardef@6f575org.highwire.dtl.DTLVardef@dcf737org.highwire.dtl.DTLVardef@ea4ec9_HPS_FORMAT_FIGEXP M_FIG C_FIG SYNOPSISEnzymatic defluorination of PFAS is rarely observed in environmental systems. This study identifies hydrolytic defluorination of branched PFOA isomers, improving understanding of PFAS defluorination at the enzyme level.

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.

Conformational dynamics play a central role in enzyme function by controlling substrate access and productive binding. Yet mutations that beneficially modulate these properties are difficult to identify. Here, we used ultrahigh-throughput fluorescence-activated droplet sorting (FADS) with a bulky fluorogenic substrate derived from coumarin (COU-3) to impose steric selection pressure on the haloalkane dehalogenase LinB. Screening a focused library yielded five single substitutions located 11.5-15.5 [A] from the catalytic centre. Variant I138N showed a fourfold increase in catalytic efficiency toward COU-3 through reduced KM and increased kcat, associated with increased cap-domain flexibility and facilitated substrate entry. In contrast, variant P208S markedly reduced substrate inhibition and shifted specificity toward bulkier iodinated haloalkanes by reshaping its tunnel environment. Integrated kinetic and structural analyses revealed that screening with bulky substrates directs selection toward distal regions controlling substrate access and unproductive binding. These findings demonstrate that ultrahigh-throughput FADS can reveal dynamic mechanisms of enzyme adaptation that remain difficult to predict by rational design. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/713925v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@782038org.highwire.dtl.DTLVardef@8b43f3org.highwire.dtl.DTLVardef@11a403eorg.highwire.dtl.DTLVardef@6fcaea_HPS_FORMAT_FIGEXP M_FIG C_FIG

Phenazines are redox-active microbial metabolites produced and secreted in diverse ecological contexts from soils to chronic infections. In these disparate environments phenazines can function variously as antibiotics, extracellular electron shuttles, and nutrient scavengers. Key to understanding the impact of these functions is a robust expectation of phenazine retention or diffusion in a given context. But predicting phenazine fate and transport is difficult because of the chemical complexity of their local microenvironments. To address this challenge, we measured the octanol water distribution coefficient (LogD) as a proxy for lipophilicity of three naturally occurring phenazines produced by the opportunistic pathogen Pseudomonas aeruginosa: phenazine-1-carboxylic acid, phenazine-1-carboxamide, and pyocyanin. We investigated the behavior of both oxidized and reduced forms of these phenazines across broad ionic strength and pH conditions. While the ionic context exerts only small effects, the pH and redox state contribute strongly and independently to changes in phenazine lipophilicity. The pH trends are expected, but the observed redox dependence is generally missed by existing lipophilicity calculation methods. Additional LogD measurements with 1-hydroxyphenazine and unsubstituted phenazine, together with density functional theory modeling of phenazines in their reduced and oxidized forms, reveal that intramolecular hydrogen bonding contributes significantly to the increased lipophilicity of reduced phenazines that possess H-bond accepting substituents in the 1-position. These results explain phenazine behavior in a biological context: redox state alone significantly alters retention of pyocyanin in planktonic P. aeruginosa cells, with the reduced species being predominantly retained by membranes. We propose that the modulation of phenazine lipophilicity in response to the local redox environment has evolved to give a competitive advantage to bacteria by retaining or dispersing these bioactive molecules. Beyond improving our understanding of natural phenazine fate in diverse microbial contexts, our results emphasize an oft-overlooked theme relevant to rational drug and electrochemical shuttle design: redox state matters.

Histamine is a key signaling molecule in pathophysiology that can exhibit significant regulatory roles in diverse health and disease status. Besides the well-studied noncovalent interactions between histamine and its receptors, protein histaminylation is a recently discovered mode of action, through which histamine regulates cellular signaling pathways in a covalent-interaction manner. Histaminylation is an emerging protein post-translational modification, where an isopeptide bond is formed between the histamine primary amine and {gamma}-carboxyl group of glutamine through a transamidation reaction catalyzed by transglutaminase 2 (TGM2). However, due to the lack of efficient pan-specific antibodies targeting histaminylated glutamine, the histaminylation proteome in cells remains poorly explored. Here, we report the design and development of a novel N{tau}-propargylated histamine probe as well as its successful application in chemical proteomic profiling of the histaminylation proteome in cancer cells. Notably, new TGM2-catalyzed epigenetic marks on core histones, e.g., H2AXQ84 and Q104 histaminylation, have been identified from cancer cells and verified in this study.

Predicting high-concentration viscosity of monoclonal antibodies such as IgG1 is crucial for their development as therapeutics for subcutaneous delivery. Unfortunately, traditional experimental rheometry methods for assessing viscosity are low-throughput. This study evaluates Self-Interaction Nanoparticle Spectroscopy (SINS) assays--specifically charge-stabilized SINS (CS-SINS) and PEG-stabilized SINS (PS-SINS)--for high-throughput viscosity prediction. We characterized 96 IgG1 antibodies, assessing SINS against in silico descriptors and dynamic light scattering (DLS) data. CS-SINS showed strong correlation with charge, offering limited additional utility. In contrast, PS-SINS provided orthogonal information; integrating it with in silico data and DLS significantly improved random forest model accuracy for binary viscosity classification. PS-SINS measurements in multiple buffers captured complementary information, achieving comparable accuracy without DLS. Importantly, PS-SINS scores exhibited a strong logarithmic relationship (r=0.98) with high-concentration viscosity in Fc variants of clinical antibodies, suggesting a direct mechanistic link. Furthermore, PS-SINS performed reliably with one column purified (protein A) samples, supporting its early-stage application. These findings establish PS-SINS as a high-throughput tool to accelerate the developability assessment of antibody candidates.

Human serum albumin (hSA) is the most abundant protein in human plasma, and its pharmacological properties, such as long plasma half-life mediated by the neonatal Fc receptor (FcRn) and its ability to bind endogenous and exogenous molecules, make it attractive for biotechnological applications. Currently, most wild type (WT) SAs are derived from human or bovine serum or produced in yeast and mammalian cells. Although well established, these methods are costly, difficult to reproduce, and not environmentally sustainable. Building on a previous study to design highly mutated hSA sequences, we extend the validation through an in-depth analysis of three engineered hSA variants; hSA1, hSA2, and hSA3, containing 16, 25, or 73 amino acid substitutions, respectively. These variants were designed for enhanced solubility, stability, and expression in Escherichia coli. All three variants showed low- micromolar affinities for hFcRn at pH 5.5, and negligible binding at pH 7.4. In a human endothelial cell-based recycling assay (HERA), the engineered hSA variants were recycled by hFcRn to the same extent as hSA isolated from serum. Exploring the properties of canonical drug-binding sites, warfarin affinity was comparable to WT hSA, whereas ibuprofen binding differed. Complementary cytotoxicity assays on human macrophages confirmed negligible toxicity and biocompatibility. A cryo-electron microscopy structure of hSA3 revealed that, despite extensive engineering, the native heart-shape of hSA, folding of domains, and its open conformation were preserved. These findings validate the structural integrity and functional adaptability of engineered hSA variants, underscoring their potential as versatile, animal-free solutions for next-generation therapeutics and biotechnological applications.

Untargeted proteomics enables quantitative determination of host cell proteins (HCPs) in biotherapeutics, yet no workflow has been validated under ICH Q2(R2) for regulated quality control. We report a prospective validation of label-free untargeted proteomics for HCP quantification using a total-error (TE) approach. A stable isotope-labeled whole-proteome standard was spiked into NISTmAb at seven levels (20-80 ng). Four independent assays (198 injections) supported hierarchical replication and one-way random-effects ANOVA variance decomposition with Welch-Satterthwaite adjustment. Dual entrapment analysis demonstrated empirical peptide-level false discovery proportions below 1% at q = 0.01. Deterministic parsimony inference ensured invariant protein-group definition. Weighted least-squares regression (R{superscript 2} = 0.993) identified stable proportional compression with recoveries of 81-85%. Repeatability dominated the variance structure (median CV 2.7%); intermediate precision total SD ranged from 0.69% to 3.81% over the validated range. Accuracy profiles integrating empirical bias with a log- log variance model showed 95% {beta}-expectation and 95/95 content tolerance intervals fully contained within {+/-}30%, with a lower limit of quantification (LLOQ) of 20 ng. Abundance-stratified TE analysis revealed concentration-dependent calibration heterogeneity masked by aggregate-level estimation; stratum-specific {beta}-expectation intervals within {+/-}35% defined an abundance-aware LLOQ of 3.6 ppm (P95 = 3.87 ppm). Robustness under independent search software (FragPipe, CCC = 0.998, LoA {+/-}9%) and cross-platform acquisition (Astral, CCC = 0.980, LoA {+/-}18%) remained within predefined {+/-}30% agreement limits. System suitability criteria were derived empirically from validation performance. This is the first prospective ICH Q2(R2)-aligned validation of untargeted proteomics for HCP quantification, with a statistical framework applicable to other high-dimensional analytical methods requiring regulatory qualification. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/710150v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1f5331aorg.highwire.dtl.DTLVardef@ee2234org.highwire.dtl.DTLVardef@798eaorg.highwire.dtl.DTLVardef@c84034_HPS_FORMAT_FIGEXP M_FIG C_FIG

The structural study of membrane proteins has traditionally relied on detergent-based extraction from cellular membranes. Although native-like reconstitution approaches have advanced, fully understanding membrane protein dynamics requires examining them within their native membrane environment. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for probing structural dynamics in reconstituted systems, but the presence of the lipid bilayer introduces considerable complexity, limiting broader adoption under physiological conditions. Here, we present the first fully automated HDX-MS platform incorporating a two-stage delipidation workflow. We applied this approach to monitor the dynamics of the ABC transporter MsbA in isolated inner membrane vesicles (IIMVs) from Escherichia coli through its ATPase cycle. IIMVs revealed distinct dynamic features within the nucleotide binding domains and substrate binding cavity, highlighting physiologically relevant motions not observed with detergent solubilised MsbA. This platform significantly advances HDX-MS and underscores the importance of studying membrane proteins in native lipid environments.

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 conformational dynamics of a model cationic protein in water and in the presence of anionic sodium dodecyl sulphate (SDS) and cationic cetyltrimethylamonium bromide (CTAB) surfactants at different concentrations were investigated using all-atom molecular dynamics simulations. Free-energy landscapes constructed along principal components reveal a compact, well-defined native basin at 25 {degrees}C in water, whereas elevated temperature (100 {degrees}C) induces a broadening of the conformational space and the emergence of multiple metastable states. The presence of surfactants further modulates this behavior in a concentration-dependent manner. Cluster population analysis shows that SDS promotes a highly heterogeneous ensemble characterized by reduced dominance of the native-like cluster, while CTAB partially protects the protein from thermal denaturation at higher concentrations. Radial distribution functions demonstrate strong accumulation of SDS headgroups around the protein and pronounced insertion of SDS alkyl tails into hydrophobic protein regions, indicating direct hydrophobic destabilization and micelle-assisted unfolding. In contrast, CTAB exhibits weaker headgroup association owing to electrostatic repulsion and reduced tail-hydrophobic contacts, suggesting a less disruptive interaction mechanism. At high concentration, CTAB aggregates provide a structured hydrophobic environment that stabilizes the folded state and suppresses denaturation. Together, these results provide a molecular-level picture of how surfactant chemistry and concentration govern the conformational stability of a cationic protein, highlighting the dominant role of hydrophobic interactions in surfactant-induced denaturation at high temperature. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/717321v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@dcf96aorg.highwire.dtl.DTLVardef@17acdc7org.highwire.dtl.DTLVardef@15bdc2borg.highwire.dtl.DTLVardef@1d39f3c_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

Accurate identification of drug target proteins remain major challenges in proteomics-based target discovery, particularly for low-abundance nuclear proteins that are difficult to detect because of the complexity of whole-cell lysates. Here, we developed a detergent-free nuclear-cytoplasmic fractionation strategy compatible with peptide-centric local stability analysis (PELSA), which markedly improves detection of nuclear drug targets. Using K562 cells, we demonstrated that mild detergent-free fractionation enables high-fidelity nuclear-cytoplasmic separation with minimal cross-contamination. When coupled with PELSA, this workflow significantly increases the number of detected nuclear targets relative to whole-cell analysis. Benchmarking with well-characterized nuclear drugs, including the histone deacetylase inhibitor panobinostat and the RNA polymerase II inhibitor -amanitin, our results showed improved identification of canonical nuclear targets. Broad profiling of staurosporine target further revealed expanded kinase target coverage by combining the results of nuclear and cytoplasmic fraction, with the CLK family kinases detected exclusively in the nuclear fractions. Additionally, we showed that PELSA can also be performed on intact nucleus level. Collectively, these findings establish detergent-free nuclear-cytoplasmic fractionation-PELSA as a robust and scalable strategy for spatially resolved drug target identification, improving sensitivity for nuclear and low-abundance proteins.

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.