Nature Chemistry

Therapeutic peptides combine high target specificity with potent biological activity.1 However, treatment success is often limited by rapid clearance and the need for frequent injections.2, 3 This challenge is particularly acute for therapeutic peptides used in obesity, where clinical benefit must be balanced against dose-dependent adverse effects. In nature, these constraints are overcome by storing hormones as reversible fibrils,4 but pharmacokinetic control is essential for widespread adoption of bio-inspired self-assembled depots for therapeutic peptides. Here, we show that tuneable pharmacokinetics can be achieved and modelled by mapping the fundamental chemical parameters of reversibly self-assembly in vitro. We demonstrate this approach for the amylin analogue pramlintide. Amylin analogues are under development for the next generation of diabetes and obesity treatments, with improved mechanism of action e.g. preserving lean body mass.5-8 Pramlintide is an approved drug with a well-established safety profile, however, it has a comparable half-life to native amylin.8-12 In a pilot study, we achieve in vitro-in vivo correlation, increasing the half-life of pramlintide 20-82-fold in rats, while controlling burst release. These findings demonstrate that the optimisation of pharmacokinetics can be decoupled from peptide engineering, establishing a generalisable framework for generating long-acting peptide formulations by emulating native storage mechanisms.

Steroid-based fluorescent-quencher probes now enable real-time, residue-level mapping of previously inaccessible cholesterol-binding sites on G-protein-coupled receptors. We designed Tide Quencher 1 (TQ1) conjugated steroids that target two distinct peripheral sites on the M1 muscarinic receptor. One near the extracellular N-terminus and another adjacent to the intracellular C-terminus. Using pregnanolone glutamate as a versatile scaffold, we synthesised a library of probes varying in C-3 linker length ({gamma}-aminobutyric acid vs. L-glutamic acid) and C-3/C-5 stereochemistry (3/3{beta}/5/5{beta}). Fluorescence-quenching assays with CFP-tagged receptors revealed that TQ1 probes consistently outperformed Dabcyl, delivering up to 40 % quenching within minutes and sub-micromolar EC50 values. The most potent N-terminal probe (35-PRG-Glu-TQ1 (5)) achieved 300 nM potency, while the best C-terminal probe (35{beta}-PRG-Glu-TQ1 (3)) reached 1 {micro}M potency with rapid association. Molecular docking and MD simulations identified key residues (K20, Q24, W405 at the N-site; K57, Y62, W150 at the C-site) mediating binding, a prediction confirmed by alanine-scan mutagenesis that markedly reduced quenching at the N-terminus and only modestly affected the C-terminus. Competition experiments with non-quenching analogues further validated probe specificity. Crucially, the pregnane core proved essential; alternative steroid backbones failed to generate robust quenching. This fluorescence-quenching platform overcomes the limitations of traditional radioligand assays, providing kinetic insight, high-throughput compatibility, and the ability to dissect lipid-GPCR interactions in native membranes. The approach is readily extensible to other GPCR families, opening new avenues for structure-guided drug discovery targeting allosteric cholesterol sites.

High-valent metal-nitrido species are powerful nitrogen-atom transfer intermediates but remain difficult to access and control due to intrinsic instability and bimolecular N-N coupling pathways. Herein, we report the first formation of a high-valent Mn(V)-nitrido complex within a de novo designed protein scaffold and demonstrate that a reactive precursor to this species can be catalytically intercepted for enantioselective aziridination. A Mn(V){equiv}N unit derived from an abiological diphenyl porphyrin is confined within a designed helical bundle protein, where the protein environment suppresses bimolecular decay and enables detailed spectroscopic characterization. Electron paramagnetic resonance, resonance Raman, and circular dichroism spectroscopies confirm formation of a low-spin Mn(V)-nitrido species that is stable for weeks at room temperature and exhibits minimal perturbation of the Mn{equiv}N unit upon modulation of the axial histidine ligand, while catalytic activity and stereochemical outcome are sensitive to its presence. Mechanistic studies identify monochloramine (NH2Cl) as the operative nitrogen-atom donor and support the involvement of a transient Mn-bound N-transfer intermediate en route to nitrido formation. Under catalytic conditions, this intermediate is inter-cepted to perform aziridination with TON {approx} 180 and an enantiomeric ratio of 65:35. Together, these results establish de novo protein design as a platform for stabilizing high-valent metal-nitrido species and harnessing their reactivity for nitrogen-atom transfer chemistry beyond the limits of natural metalloenzymes and small-molecule catalysts.

Direct solar-to-chemical conversion offers a compelling route to clean, dispatchable energy. Photosystem I (PSI), an evolutionarily optimized light-driven oxidoreductase central to oxygenic photosynthesis, can be repurposed for direct solar-fuel production by efficiently coupling its photochemistry to catalysts, thereby storing sunlight as chemical energy in the H-H bond of H2. One promising architecture integrates PSI with Pt nanoparticle (PtNP) catalysts to create photocatalytic PSI-PtNP biohybrids. Advancing these systems requires molecular-level insight into protein-nanoparticle interactions and the bio-nano electron transfer pathways that govern activity; however, progress has been constrained by limited structural data to guide rational design. Here, we present two molecular structures of active PSI-PtNP assemblies that (a) compare thermophilic and mesophilic PSI scaffolds and (b) probe how removal of the terminal [4Fe-4S] clusters and stromal subunits in PSI reshapes protein-nanoparticle interfaces and photocatalysis. Structural analyses and molecular dynamics simulations define the interface topology, electrostatics, and cofactor-to-nanoparticle distances, revealing key molecular features that control biohybrid formation and electron transfer efficiency. These data establish mechanistic links between scaffold composition, bio-nano interface geometry, and catalytic performance, yielding design principles for optimizing PSI-PtNP architectures. The resulting structure-function insights provide a blueprint for engineering PSI-based solar-fuels systems and, more broadly, inform the design of protein-nanomaterial interfaces for light-driven catalysis.

Life is predicated on chirality, a molecular asymmetry akin to the left and right versions of human hands. Here we show that privileged protein residues are predisposed for chiral regulation. We developed enantiomeric oxaziridine reagents that systematically identify pro-(S) and pro-(R) methionine oxidation sites across proteomes that can be erased by stereospecific methionine sulfoxide reductase enzymes A and B, respectively. These probes reveal that chiral regulation of methionine oxidation-reduction processes can allosterically regulate protein function, as shown in cell and murine models of oxidative stress where selective (R)-methionine sulfoxide formation on M69 of biphenyl hydrolase-like protein leads to hydrolase inhibition and amplification of proteome N-homocysteinylation modifications. This work introduces a platform for characterizing sites of asymmetric methionine oxidation and the functional consequences concomitant with an individual chiral single-atom modification.

Cells utilize liquid-liquid phase separation to organize biochemical reactions within biomolecular condensates, which function as membraneless organelles. Although these assemblies are known to enhance reaction rates by concentrating reactants, the mechanisms beyond simple mass-action effects remain poorly understood. Here, we examined how the physicochemical microenvironment within condensates modulates reaction kinetics using spontaneous protein ligation as a model reaction, conducting a systematic analysis across various condensates, ranging from structured scaffolds (PRM-SH3 systems) to intrinsically disordered protein (IDP)-based scaffolds such as LAF, TAF, and FUS. We designed a FRET-based proximity-sensitive client probe to quantify increases in effective local concentration arising from excluded-volume effects. In parallel, we measured internal hydrophilicity and water activity, revealing them as additional key determinants of reaction acceleration. Together, the findings presented here elucidate how phase-separated compartments regulate biochemical reactions through the interplay of physical (effective concentration) and chemical (hydrophilicity and water activity) microenvironments and provide mechanistic insights for engineering condensates with tunable reactivity.

Protein self-assembly enables precise nanoscale organization but rarely translates into macroscopic materials that retain functionality beyond aqueous environments. Here, we report that a bacterial microcompartment (BMC) trimer fused with SpyTag (T1-SpyTag), when expressed as a standalone component, undergoes rapid and spontaneous self-assembly into macroscopically visible fibers and layered sheets. These structures span from the nanoscale to the millimeter scale, forming robust three-dimensional protein materials that remain structurally intact and functionally accessible in both solution and dried states. Unlike previously reported SpyTag-enabled BMC systems that function primarily as passive cargo-loading modules, T1-SpyTag macromolecular structures exhibit emergent material behavior, including chemical robustness under denaturing conditions, while preserving covalent reactivity toward SpyCatcher-fused cargos. The multilayered architecture enables tunable surface display, access to ultrathin, processable protein films, and surface renewability through layer-by-layer removal and regeneration. This work demonstrates how a minimal genetic modification of a native protein building block can drive the formation of functional, macroscopic protein materials, thus expanding the design space of BMC-derived assemblies for biointerfaces, catalysis, and sustainable protein materials engineering.

Bile salt hydrolases (BSHs) are gut microbial enzymes that catalyze the deconjugation of glycine-or taurine-conjugated bile acids (BAs), a key step in shaping the BA pool in the human gastrointestinal tract and modulating host-gut microbiome interactions.1-3 All known BSHs are members of the N-terminal nucleophile (Ntn) hydrolase superfamily and share a conserved architecture and mechanism involving a nucleophilic active site cysteine.4,5 This knowledge has guided predictions and study of BSH activity in the gut microbiome6,7 as well as the development of BSH inhibitors8. Here, we report the discovery and characterization of a previously unknown BSH from the human gut bacterium Bilophila wadsworthia that belongs to the metal-dependent amidohydrolase superfamily and exhibits robust and specific activity toward taurine-conjugated bile salts. We show this secreted enzyme, metalloBSH, utilizes a metallocofactor for BA deconjugation, a mechanism distinct from that of canonical Ntn-type BSHs. MetalloBSHs are conserved in B. wadsworthia and present in many other Desulfovibrionaceae found in vertebrate gut microbiomes. Analysis of multi-omic datasets indicates metalloBSHs are expressed in vivo and correlate with BA metabolism. Overall, our findings reshape our understanding of BSH activity in the gut microbiome and highlight the promise of activity guided discovery in revealing previously overlooked gut microbial enzymes.

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

Insulin glargine is formulated at acidic pH but acts after transferring to near-neutral tissue, where its prolonged effect is commonly attributed to isoelectric depot formation. However, the structural pathway linking precipitation to delayed release has remained unresolved. Here we combine ambient-temperature serial femtosecond crystallography, solution biophysics, and multiscale network analyses to define the pH-dependent conformational landscape of hexameric glargine across pH 8.4, 7.3, 6.4, and 5.1. We resolve full hexameric glargine structures and identify a previously unreported, pH-coupled lattice transition from P1211 (near-neutral) to R3:H (acidic), accompanied by redistribution from compact phenolic Rf6-state assemblies to more plastic yet structurally coherent TRf/T3Rf3 states. This transition is accompanied by B-chain N-terminal unpeeling, phenol-pocket collapse, hydration loss, and electrostatic rewiring, and is mirrored in solution by oligomeric heterogeneity, Raman amide-I broadening, reduced thermal stability, and a blue-shifted intrinsic fluorescence maximum. Multiscale analyses further indicate that acidification does not create a new dynamical regime but reweighs pre-existing collective modes along a continuous free-energy landscape. These results support a revised mechanism in which isoelectric precipitation and delayed dissociation are mechanistically coupled through structurally organized molten-like intermediate states, linking glargine pharmacology to intrinsic allosteric redistribution within the hexamer. These findings establish a structural blueprint for benchmarking biosimilar glargine and for engineering next-generation basal insulins by tuning allosteric plasticity and intermediate-state stability.

Apolipoprotein A-I mimetic 4F, an 18-residue amphipathic -helix, can self-assemble with lipids to form peptide nanodiscs, yet the molecular determinants governing their assembly and stability remain poorly understood. Here, using coarse-grained molecular dynamics (CG-MD), we capture the de novo formation of 4F nanodiscs with DMPC and reveal a multistep assembly pathway involving nucleation, fusion, and ellipse-to-disc maturation. All-atom back-mapping shows that the nanodisc rim is structurally heterogeneous and stabilized by aromatic-acyl interactions, Lys/Arg headgroup anchoring, and inter-peptide electrostatic contacts. Lipid composition and temperature critically regulate nanodisc integrity: DMPC supports continuous peptide belts and long-term stability, whereas DPPC below its main phase transition temperature suppresses fusion and yields fragmented, non-uniform rims. These findings validate the ability of CG-MD to resolve nanodisc assembly mechanisms. Experimental measurements corroborate the simulations, demonstrating that 4F nanodiscs exhibit lower thermal resilience than MSP nanodiscs while retaining structural integrity at moderate temperatures. As a functional benchmark, MSP nanodiscs inhibit amyloidogenic A{beta} fibrillization, consistent with our earlier findings for 4F nanodiscs, indicating that a peptide-rimmed discoidal architecture is sufficient to suppress amyloid nucleation. Together, these results establish a mechanistic framework and design principles for single-helix peptide nanodiscs and delineate the conditions under which they converge with or diverge from MSP-based scaffolds.

Adenosine 5-triphosphate (ATP) is found to form biomolecular condensates with proteins. However, without complementary proteins, the small size and high charge density of ATP molecules create substantial electrostatic and entropic barriers that prevent them from forming condensates. Here, we find that macromolecular crowding overcomes these energetic barriers, promoting ATP molecules to self-associate and form protein-free liquid-like condensates through screened electrostatic repulsion and enhanced hydrogen bonding. Importantly, ATP condensates are responsive to multiple stimuli and create distinct microenvironments that selectively enrich various guest molecules and protect ribonucleic acids from DNAzyme cleavage. These findings uncover important roles of ATP in forming dynamic, chemically distinct condensates via homotypic interactions, potentially expanding its classical view beyond a canonical energy carrier to a structural and regulatory architect in cellular physiology and prebiotic chemistry.

Adenine is a ubiquitous nucleobase found in nucleic acids, cofactors, and signaling molecules and mediates diverse molecular interactions. Here, we identify TvAPT, an adenine prenyltransferase from the cyanobacterium Trichormus variabilis NIES-23. Unlike canonical enzymes limited to C5 dimethylallylation, TvAPT efficiently catalyzes the unprecedented N6-prenylation of adenine-containing substrates using extended prenyl donors (C10 and C15), markedly increasing the hydrophobicity of the adenine moiety. X-ray structural analysis and protein engineering revealed that an enlarged prenyl-binding pocket enables this donor promiscuity, allowing rational tuning of prenyl-donor preference. These findings establish TvAPT as a versatile biocatalytic platform that expands the chemical space of adenine-containing molecules for biomolecular engineering, as demonstrated by the synthesis of membrane-permeable nucleotides and analogues of plant signaling molecules.

Multicomponent Rieske oxygenases catalyze diverse oxidative transformations but require precisely matched redox partners to sustain efficient electron transfer, severely limiting their modularity and biocatalytic application. Yet, the molecular logic underlying this specificity remains poorly defined. Here we decode the molecular principles governing redox partner specificity in representative three-component Rieske oxygenase systems. Through systematic mutagenesis analysis and cross-component reconstitution assays, we identify a single ferredoxin residue that acts as a class-defining determinant of oxygenase recognition. Guided by this insight, we reprogram electron transfer between non-cognate components by complementary engineering of the oxygenase interface, creating an unnatural redox chain with substantially enhanced catalytic turnover compared to the native system. Spectroscopic, binding and computational analyses reveal that productive electron transfer arises from optimized electrostatic complementarity and redox potential alignment rather than maximal binding affinity. Extending this strategy to another oxygenase system demonstrates its generality. Together, these results establish transferable design rules for rationally engineering electron transfer pathways in multicomponent oxygenases, enabling their predictable adaptation as customizable biocatalysts.

Lasso peptides adopt a distinctive [1]rotaxane conformation, yet the principles governing the folding of this kinetically trapped structure have remained elusive. Here, we integrated extensive molecular dynamics simulations and deep learning to elucidate the de novo folding mechanism of 20 lasso peptides lacking secondary post-translational modifications. We constructed Multi-Ensemble Markov Models for each lasso peptide and uncovered a universal uphill folding landscape with spontaneous folding probabilities consistently below 0.8%. Loop stability strongly correlated with folding propensity, and targeted experiments further validated that enhancing loop {beta}-hairpin formation promotes folding of microcin J25, the well-studied lasso peptide extensively characterized as an in vitro model. Additionally, the substantial entropy cost opposed lasso peptide folding. Simulations mimicking enzymatic spatial confinement reduced this penalty and stabilize folding. Leveraging Variational AutoEncoder-based pathway clustering, we resolved distinct pathway channels and representative folding pathways. Together, these findings establish representative folding models and fundamental thermodynamic and kinetic principles for rational engineering of lasso peptides.

Antimicrobial peptides (AMPs) that also form functional amyloids exhibit remarkable environmental sensitivity, yet the physicochemical rules governing their structural switching remain unresolved. Here, we investigate how surfactant charge and assembly dynamics regulate the antimicrobial-amyloidogenic transition of Uperin 3.5, a 17-residue amphibian AMP with pronounced conformational plasticity. Using an integrated approach combining all-atom molecular dynamics simulations with circular dichroism and thioflavin T fluorescence assays, we systematically probe the effects of surfactant identity, concentration relative to the critical micelle concentration (CMC), peptide stoichiometry and ionic strength. We show that -helical stabilisation and antimicrobial-like behaviour scale directly with surfactant charge: anionic Sodium dodecyl sulphate (SDS) induces the highest helicity in monomeric Uperin 3.5 ({approx}80-90%), followed by zwitterionic dodecyl-phosphocholine (DPC) ({approx}35-45%), while cationic Cetyltrimethylammonium bromide (CTAB) fails to stabilise secondary structure. This charge-ordered trend is mirrored in oligomer remodelling, with SDS driving the most rapid dissociation of {beta}-sheet tetramers, DPC inducing slower partial disassembly and CTAB exhibiting minimal effect. Above the CMC, micellar environments stabilise amphipathic -helical states and efficiently dissolve amyloid assemblies. In striking contrast, under below-CMC conditions, limited SDS availability combined with peptide crowding promotes cooperative aggregation, where surfactant monomers act as dynamic scaffolds that nucleate N-terminal {beta}-sheet interactions--an effect strongly accelerated by physiological salt. Large-scale simulations reveal mixed /{beta} aggregates whose formation is governed by electrostatic screening and surfactant-mediated co-assembly. Together, these findings establish surfactant charge and assembly state as quantitative, environment-dependent regulators of functional amyloidogenesis in antimicrobial peptides. More broadly, they suggest that controlled modulation of membrane-mimetic environments can be exploited to bias peptides toward antimicrobial or amyloidogenic states, offering conceptual avenues for therapeutic strategies targeting peptide misfolding and neurodegenerative disorders.

O_LIPlant growth is influenced by the composition of its associated microbiome. The inherent complexity and functional redundancy of natural plant microbiomes presents a formidable barrier to understanding the myriad biological interactions therein. Efforts have been made to develop synthetic microbial communities (SynComs) that can provide a rigorous and generalizable framework for the rational design of next-generation microbial products for sustainable agriculture. We test multiple strategies for stable, plant growth promoting SynCom design and evaluate the phenotypic and molecular impacts of a successful plant-SynCom interaction. C_LIO_LIWe designed 4 distinct, reduced-complexity variants of SynCom SRC1 and assessed their capacities for colonization, stability, and plant growth promotion. To understand the impact on plant performance of our highest performing SynCom variant, we characterized the hosts longitudinal transcriptional response to SynCom inoculation and corroborated the results with metabolomics analysis. C_LIO_LIThe top performing SynCom stably colonized sorghum roots and rhizospheres, elicited plant growth promotion, and induced dynamic spatiotemporal gene transcription in sorghum roots and shoots defined by modulation of growth-defense tradeoff machinery and enhanced flavonoid production. C_LIO_LIThe resultant reduced-complexity SynCom is a highly stable, soil-independent, plant growth promoting, and demonstrates the utility of colonization-based selection criteria, integrated with longitudinal transcriptomic and metabolomic characterization. C_LI

The plant plasma membrane is a highly dynamic structure that is crucial for cell compartmentalization, the maintenance of (bio)chemical gradients, signaling and cell growth and responses to stress. In plants, plasma membranes are tightly connected to the cell walls that encase them. These cell walls can act as diffusion barriers and prevent the use of a wide range of synthetic fluorescent probes that have been developed to study animal cell membranes, which lack a cell wall, with live functional imaging. Here, we introduce LipoTag, a minimal chemical motif that, upon chemical conjugation, transforms hydrophobic fluorophores into water-soluble, membrane-targeted probes that can permeate plant cell walls to reach their intended location. LipoTag uses a localized positive charge in combination with a short aliphatic spacer to direct cargo to the plasma membrane. We used LipoTag to develop a suite of membrane-specific fluorescent probes that work in walled organisms beyond the plant kingdom. In addition, we used LipoTag to develop functional reporters for the quantitative imaging of membrane density, lipid order and membrane oxidation in living plant tissues. LipoTag forms a modular platform for exploring the plant plasma membrane with a suite of contemporary imaging modalities.

Proteins with intrinsically disordered regions (IDRs) perform essential cellular functions despite lacking stable structures, challenging the traditional structure-function paradigm. Neurofilament-light (NFL) proteins assemble into bottlebrush filaments, whose disordered tail domains mediate nematic hydrogel formation critical for neuronal integrity. Mutations in NFL are linked to Charcot-Marie-Tooth (CMT) disease, yet their molecular effects remain unclear. Here, aiming to gain insight into these molecular mechanisms, we combine small-angle X-ray scattering, microscopy, and deep-learning conformational analysis to investigate CMT-associated NFL tail mutations. We find that these mutations induce pathological hydrogel compaction, disrupt filament nematic order by generating microdomains, and alter water retention dynamics by reshaping of sequence-dependent conformational ensembles, leading to macroscopic network rearrangements. These findings provide mechanistic insight into how subtle sequence changes in IDRs modulate protein network organization and function, informing an understanding of IDR-related pathologies and mutation-based disease characterization.

Prion diseases are a group of fatal neurodegenerative diseases that proceed through the templated conversion of the normal PrPC protein to a self-propagating and infectious form, termed PrPSc. This conversion process is central to disease progression. However, due to difficulties in producing functional PrPSc molecules that can be selectively modified with chemical probes, many aspects of PrPSc biology cannot be directly studied. To overcome this limitation, we substituted p-azido-L-phenylalanine (AzF), a small click chemistry-reactive amino acid, for tryptophan residue 99 of PrPC. W99AzF PrPC substrate can efficiently and faithfully propagate either infectious or non-infectious PrPSc conformers in vitro. Critically, W99AzF PrPSc amyloid fibrils remain amenable to click chemistry by various ligands after the prion conversion process. Through the combination of site-specific substitution, the modularity of click chemistry, and the functional diversity of click labels, a multitude of modified prions can now be produced to ask targeted questions about the biochemical and biological basis of prion infectivity. O_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC="FIGDIR/small/717205v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1c5a978org.highwire.dtl.DTLVardef@1f93e9corg.highwire.dtl.DTLVardef@7d8116org.highwire.dtl.DTLVardef@1a5e95a_HPS_FORMAT_FIGEXP M_FIG C_FIG