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.

On-demand assembly of arbitrary, sequence-defined polymers from a pool of monomers is a major challenge in modern chemistry, towards which limited progress has been made. By contrast, biological systems routinely use information-bearing DNA and RNA templates to catalytically synthesize a precise, far-from-equilibrium ensemble of nucleic acid and protein sequences from the available pools of NTPs or aminoacyl-tRNAs. Inspired by these biological examples, we introduce an enzyme-free DNA strand displacement network in which single-stranded DNA sequences template the assembly of specific non-covalent DNA multimers of up to length five, under isothermal and autonomous conditions. The templates demonstrate significant turnover, bypassing product inhibition. They can thereby catalyse the formation of a far-from-equilibrium ensemble of long-lived metastable products that are not otherwise addressable.

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.

Antibody and protein-drug conjugates (XDCs) have emerged as promising cancer therapeutics, yet their clinical utility remains constrained by dose-limiting toxicities and narrow therapeutic windows. These safety challenges stem primarily from two factors: premature payload release during systemic circulation, and poor physicochemical properties inherent to the hydrophobic cytotoxic drugs they carry. Prior strategies attempted to address these limitations by appending water-soluble tags to reduce overall conjugate hydrophobicity, but achieved only modest improvements. As a result, the hydrophobic nature of cytotoxic payloads has remained a persistent obstacle in XDC development. Here, we report a fundamentally different chemical strategy that reframes this liability as a design opportunity. Rather than masking drug hydrophobicity, we exploit it as the driving force for self-assembly of facially amphiphilic protein-drug conjugates with programmable drug moieties (PDCs). In this architecture, the hydrophobic cytotoxic drug and the hydrophilic protein serve as the core and shell, respectively, spontaneously assembling into monodisperse, well-defined spherical protein nanotherapeutics of controlled size. This design principle transforms a longstanding physicochemical challenge into a functional engineering tool, enabling precise nanostructure formation without sacrificing potency. In vitro studies confirm that the resulting nanotherapeutics effectively kill cancer cells, establishing a strong foundation for further therapeutic development.

Sinefungin is a potent nucleoside antimetabolite of S-adenosylmethionine (SAM), yet its biosynthesis has remained unclear for decades. Here we detail the identification and characterization of the complete sinefungin biosynthetic gene cluster (BGC) from Streptomyces incarnatus NRRL 8089. In vitro and in vivo analyses demonstrate that the defining carbon-carbon (C-C) bond is formed not by the long-hypothesized PLP-dependent process, but by a vitamin B12-dependent radical SAM enzyme. Using isotope-labeled cofactors and substrates, we provide evidence that the adenosyl group of sinefungin atypically originates from adenosylcobalamin via a homolytic SH2 substitution, establishing a rare instance where adenosylcobalamin is enzymatically consumed during the reaction. Furthermore, the pathway utilizes a cryptic phosphorylation-dephosphorylation strategy to control intermediate processing and substrate recognition. We also characterize two peptide aminoacyl-tRNA ligases (PEARLs) that append alanines onto the nucleoside scaffold using tRNA-activated amino acids. The PEARLs act directly on small molecules rather than macromolecular substrates, with one PEARL capable of iterative elongation. Finally, we leverage these enzymes in a reduced multi-enzyme cascade to biosynthesize sinefungin. Together, these findings redefine radical-mediated C-C bond formation and pearlin enzyme versatility, unlocking biocatalytic possibilities to produce amino acid-nucleoside conjugates. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/726688v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@10e48deorg.highwire.dtl.DTLVardef@d220ceorg.highwire.dtl.DTLVardef@167e60borg.highwire.dtl.DTLVardef@2fddec_HPS_FORMAT_FIGEXP M_FIG C_FIG

Modern protein design methods based on deep learning allow generation of customized protein scaffolds with diverse geometries and functionalities. Here, we capitalize on these recent advances to develop hyper-thermostable de novo CO2 reductases featuring a cobalt porphyrin IX cofactor (CoPPIX). CoPPIX containing enzymes were assembled in vivo through media supplementation with cobalt salts and assessed for photocatalytic CO2 reductase activity. We identified two cysteine-ligated designs that exhibit high activity (>1000 turnovers at rates of up to 25 min-1) while suppressing competing hydrogen evolution pathways. A 2.1 [A] crystal structure shows close agreement to the design model with the Co-Cys bond programmed as intended. This study showcases the power of computational protein design in developing artificial enzymes to activate challenging molecules such as CO2.

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.

Biomolecular condensates organize cellular biochemistry, yet the principles governing their internal solvent architectures remain poorly understood. Most current models focus on macromolecular scaffolds while treating the solvent as a passive, spatially uniform background. Here, we introduce Condensate Spatial Topography via Emission Lifetimes (ConSTEL) to map the continuous solvent polarity landscape inside biomolecular condensates. Using PopZ as a model system, we show that the condensate interior contains a persistent, tunable mosaic of aqueous environments whose apparent polarity, reported by Nile Red fluorescence lifetimes, is organized by thermodynamic state and chemical cues. This microphase-separated solvent architecture defines distinct mesoscale rheological regimes, with intermediate aqueous niches supporting fast, confined tracer motion and highly polar or non-polar extremes forming a slower, viscoelastic mesh. We further demonstrate that drug-like small molecules partition non-uniformly across this landscape according to their physicochemical properties, and that exceeding local solubility limits drives "reciprocal sculpting", in which mismatched guests remodel the host solvent architecture. Together, these results highlight internal solvent organization as an active, tunable determinant of condensate material properties, molecular transport, and partitioning, and suggest that predictive models of condensate function and pharmacology would benefit from incorporating the spatial arrangement of solvent environments alongside bulk composition.

The adenylate cyclase toxin (CyaA) from Bordetella pertussis intoxicates host cells by directly translocating its N-terminal catalytic domain across the plasma membrane; however, the forces driving this unique process remain poorly defined. Here, we dissect the membrane translocation mechanisms of two peptide segments derived from CyaA: P233 and P454 from the catalytic domain and the translocation region, respectively. Both P454 and P233 are calmodulin-binding segments that are sequentially involved in the translocation and activation of the catalytic domain. Using a newly developed Droplet Interface Bilayer (DIB) approach, called DIB-Pipette, which enables direct visualization of peptide transport under controlled membrane potentials, we show that P454 translocates across membranes independently of membrane potential, whereas P233 translocation requires a negative electric membrane potential. Strikingly, covalent coupling of P233 and P454 enables efficient translocation of the resulting peptide even in the absence of a membrane potential. Together, these results suggest that two distinct membrane-active segments within CyaA act cooperatively to promote translocation at the peptide level, revealing an intrinsic mechanism that may contribute to membrane potential-dependent translocation. These findings provide new mechanistic insights into CyaA cell intoxication process and reveal a multifunctional strategy for protein delivery across membranes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/716334v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@750ab3org.highwire.dtl.DTLVardef@11a980org.highwire.dtl.DTLVardef@18f2b27org.highwire.dtl.DTLVardef@5a3a59_HPS_FORMAT_FIGEXP M_FIG C_FIG

Arylmalonate decarboxylase (AMDase) stereoselectively converts disubstituted malonates to chiral carboxylic acids, but its substrate spectrum is very limited regarding the size of the smaller substituent. Inspired by the observation that (S)-selective AMDase variants also convert larger substrates, we unlocked the synthesis of the (R)-enantiomers of -aryl and -alkenyl n-butanoic and n-pentanoic acids, respectively, in exquisite enantiopurity.

Nitrile-containing natural products are produced in all kingdoms of life. Despite the wide application of nitrile-containing peptide scaffolds in medicinal chemistry, the presence of the nitrile group is unprecedented in ribosomally synthesized and post-translationally modified peptides (RiPPs). In this work, we report the identification and characterization of a RiPP biosynthetic gene cluster (BGC), where an asparagine synthetase-like (AS-like) protein encoded in the BGC converts the C-terminal carboxylate of the precursor peptide to a nitrile. Furthermore, a multinuclear nonheme iron-dependent oxidative enzyme (MNIO) and an -ketoglutarate-dependent HExxH motif-containing enzyme (KG-HExxH) perform stereoselective {beta}-hydroxylation of aspartate and proline residues, respectively. The final product is a cysteine protease inhibitor and shows that Nature makes similar warheads as found in synthetic therapeutics such as the active ingredient of Paxlovid. These findings extend our understanding of the structural and functional diversity of RiPPs.

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.

Biomolecular condensates, often formed through liquid-liquid phase separation, are dynamic cellular compartments. Here, we demonstrate that a wide range of fluorescently tagged proteins undergo inadvertent, condensate-mediated crosslinking, resulting in rapid solidification of condensates under common fluorescence imaging conditions. The process is driven by excitation-induced, short-lived reactive oxygen species (ROS), whose otherwise limited crosslinking potential becomes uniquely enabled in the dense phase. In live cells, excitation-induced ROS potently trigger stress granule formation, while the ROS-driven solidification of condensates is modulated by compartment-dependent antioxidant buffering. Our findings demonstrate that condensates create a distinct environment that enables ROS chemistry unlikely to occur in the bulk cytosol. Furthermore, the cellular redox level can be a general regulator of condensate rheology. Beyond biological insights, our findings underscore the need for scrutiny when examining fluorophore-labeled condensates.

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.

Reversibly photoswitchable fluorophores are widely used in advanced bioimaging but their design remains demanding. Here, we introduce a new series spanning the whole visible range, which results from combining a large set of fluorogens with the FAST protein scaffold. We first demonstrate that these well-established labeling fluorescent protein tags turn into negative reversible photoswitchers upon decreasing the fluorogen concentration and increasing light intensity. We then show that using not anymore one but two fluorogens adds new responses to illumination. Thus, we obtain positive reversible photoswitchers, that increase their brightness under illumination. We also generate a palette of non-covalent reversibly photoconvertible fluorescent proteins changing their fluorescence color upon illumination, a reversible behavior that still remains absent in regular fluorescent proteins. This light-induced color change opens the possibility to discriminate six spectrally similar FAST variants in live cells upon demonstrating the superiority of using multiple spectral channels for exploiting the time dependence of the fluorescence response to illumination.

Phase separation of proteins and nucleic acids (NAs) into nano-to-microscale condensates can regulate biochemical processes, including assembly and organization of cytoskeletal networks such as actin and microtubules. This study examines the functional role of condensate material properties in microtubule assembly. Learning from the sequence grammar of naturally occurring intrinsically disordered regions in microtubule-associated proteins, two-component peptide-NA condensates with programmable material properties were designed. These synthetic condensates catalyze tubulin polymerization into microtubule filaments with tunable outcomes. Tubulin preferentially partitions to the condensate interface and nucleates microtubule assembly. Enhanced tubulin self-assembly produces long filaments that exhibit branching and bundling. Using a minimal stochastic chemo-mechanical model, we show that sequence-encoded condensate viscoelasticity is a tunable element that controls filament morphologies and identifies interfacial rheology as the key regulator of filament growth. Fluorescence recovery after photobleaching experiments support this model, revealing a direct correlation between interfacial tubulin mobility and condensate-directed microtubule assembly. Distinct regimes emerge due to competition between bulk adsorption and lateral diffusion of tubulin at the condensate interface, which determines whether filament tips grow or stall. Since dynamic microtubule assembly and restructuring are essential for various cellular functions, this work highlights a critical role of condensate interfacial rheology in cytoskeletal organization.

Sea slugs in the Sacoglossa superorder are some of the few animals capable of photosynthesising by isolating and maintaining functional chloroplasts within their body1,2. While this ability allows some species in this superorder, such as Elysia viridis, to appear green, camouflaging themselves within their surroundings3,4, this species is marked by extremely bright, coloured regions. Here, we show that these animals produce a yet undiscovered class of photonic structure consisting of intracellular mixed amorphous CaCO3 and calcite spherical nanoparticles organised in non-closed-packed face-centred cubic (FCC) lattices and photonic glasses5. By mapping the distribution of the cells containing such architectures, we suggest that their colour is linked both to their function and to their biological formation via the animals renal system. Using a combination of different optical methods and cryo-electron microscopy, we reveal that the biomineralisation pathway proceeds through stages of calcium ion concentration in the kidney, transport via internal vessels, and precipitation from a dense liquid-like precursor, culminating in the formation of monodisperse nanoparticles, which are the building blocks of these photonic structures.

Crystalline inclusion proteins CipA and CipB from Photorhabdus luminescens serve as versatile scaffolds for clustering genetically fused heterologous enzymes into crystalline inclusion bodies. Although engineered Cip crystals are known to function as solid biocatalysts for improving metabolite production in bacterial cells, the phase separation behavior of Cip proteins in non-bacterial cellular environments, as well as their biochemical attributes in a soluble, non-crystalline state, remain poorly understood. This study demonstrates that CipA and CipB efficiently undergo crystallization in the cytosol of human embryonic kidney cells both at normal and hypothermic cell culture conditions. Within 72 hours post-transfection, CipA and CipB become the most abundant proteins in transfected cells and produce distinctive cytosolic crystals often exceeding 10 m at least in one of the dimensions. Co-expression of CipA and CipB drives spontaneous demixing into two distinct crystal populations, and the orthogonality is maintained even when an unrelated third protein crystallizes in the same cytosol, permitting three crystal types to coexist simultaneously. Intracellular crystals are readily isolable from cells, and once purified, these crystals are stable under physiological pH conditions. However, CipA and CipB show notable differences in their crystal dissolution kinetics and protein oligomerization states when solubilized under acidic or alkaline conditions. These findings suggest that CipA/CipB forms a robust orthogonal self-assembly pair and establish CipA/CipB crystals as an efficient platform for producing biochemically programmable intracellular crystals. These properties should extend the Cip-based scaffolding approach to mammalian cell systems for synthetic biology applications.