Nature Chemistry

Dynamic biomolecular condensates play crucial roles in intracellular compartmentalization and physiological functions. While engineering tools for compartmentalization have expanded add-on functionalities, directly amplifying the inherent catalytic machinery within biological phase-separated droplets has remained elusive. Herein, we developed a phase-separated oxidative folding reaction chamber based on protein disulfide isomerase A6 (PDIA6) by chemically targeting its active site CxxC motif to enhance enzymatic activity within PDIA6 droplets. A para-substituted N-methylated pyridinylmethanethiol (pMePySH) enhanced the catalytic oxidative folding of bovine pancreatic trypsin inhibitor, proinsulin, and antibody up to 12-fold within in vitro PDIA6 droplets. Furthermore, pMePySH targeted PDIA6 foci within the endoplasmic reticulum, significantly promoting insulin secretion. These findings offer a powerful platform for the spatiotemporal manipulation of protein folding, with profound implications for the scalable manufacturing of therapeutic antibodies and other complex biopharmaceuticals.

Phosphorylation is a major regulator of biomolecular condensation, yet it remains unclear whether clustered phosphoserines can directly tune phase behavior via metal-ion coordination. Here, using solution NMR spectroscopy and human heterochromatin protein 1 (HP1) as a model system, we show that stepwise phosphorylation of its N-terminal serine cluster generates a dynamic metal-responsive module that engages Mg{superscript 2}, Ca{superscript 2}, and Mn{superscript 2}, whereas the unmodified protein shows little or no response. Metal coordination lowers the saturation concentration of phosphorylated HP1, reshapes the temperature-dependent stability of its condensates, and modulates the effects of peptide regulators in an ion-specific manner. Our data support a model in which weak, transient metal-mediated contacts enhance intermolecular connectivity between phosphorylated HP1 molecules, promoting reversible condensation alongside canonical electrostatic interactions. These findings establish clustered phosphoserines as sequence-encoded metal-responsive elements that couple post-translational modification to the material properties of biomolecular condensates.

The emergence of respiratory, photosynthetic, and assimilatory complexes in evolution required proteins capable of binding multiple catalytic and electron-transfer cofactors while exerting fine control over their spatial arrangement. Across natural systems these cofactors are preferentially positioned in loop regions. In contrast, most protein design strategies have focused on installing cofactor-binding sites within helical elements. Here we show that introducing only a pair of appropriately placed histidine ligands into the interhelical loop regions of a canonical single-chain four-helix bundle is sufficient to create new well-defined high affinity heterocofactor binding sites. This simple modification enables the self-assembly of complexes containing up to three distinct cofactors in a single designed domain with positional specificity. Using this strategy, we creat-ed constructs containing one or two hemes in combination with Zn(II) phthalocyanine monosulfonate, Zn-heme, and the light-harvesting Zn(II) tetraphenylporphyrin tetrasulfonate. Fluorescence measurements of constructs containing the latter show efficient energy transfer between photoactive donor cofactors. By demonstrating that loop-embedded ligands support robust, modular, and evolutionarily plausible cofactor recruitment, this work provides a mechanistic explanation for the widespread placement of redox and catalytic cofactors in loops in natural proteins: only limited packing complementarity is needed, meaning that just a few mutations can introduce a functional cofactor binding site, after which additional mutations can tune affinity, reactivity, and specificity. More importantly, it establishes a straightforward path toward constructing func-tional protein domains that mirror the complexity of biological energy-conversion architectures.

Messenger RNA (mRNA)-based therapeutics have emerged as a new class of biological medicines, clearly exemplified by the global deployment of mRNA vaccines against the COVID-19 pandemic. Currently, therapeutic mRNA is primarily produced through in vitro transcription that suffers high production costs. Until now, intracellular manufacture of mRNA has been challenging due to the presence of ubiquitous RNases in vivo. Here, we have developed a new approach that protects eukaryotic mRNA from RNase degradation ensuring longevity and integrity of mRNA inside microbial cells. Through targeted strain and molecular engineering, our approach involves specially designed inserts in mRNA that facilitate formation of stabilized and protected protein-mRNA complexes. In addition to vastly improved stability, the protein-mRNA complexes enable convenient purification of mRNA from cell lysate with high purity using conventional chromatography. The work reported here promises a scalable, rapid, and low-cost approach to produce fully functional eukaryotic mRNA using well-known microbial systems.

Biological systems operate through complex molecular networks programmed by genetic information; however, constructing artificial systems with multilayered control remains a significant challenge. Here, we report a simple and integrated master-key system governed by lock DNA and master key DNA, reversibly switching diverse downstream processes ON/OFF and achieving dynamic cross-talks among distinct molecular components. The system utilizes cationic copolymer chaperones as control nodes, based on poly(L-lysine) or poly(allylamine) grafted with hydrophilic side chains, with a peptide nucleic acid (PNA) plug-in that grants sequence-specificity. We demonstrated two proof-of-concept systems: a nucleic acid-based catalytic network responsive to microRNA let-7b and a peptide-mediated transformation of lipid bilayers from two-dimensional sheets to three-dimensional vesicles. Both systems exhibited precise, modular, and programmable control with high robustness, mimicking the governing role of nucleic acids in biological systems. This strategy provides a versatile design framework for constructing biomimetic molecular networks and studying biological systems.

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.

The non-amyloid {beta} component (NAC) domain of alpha synuclein (Syn) drives Syn aggregation in Parkinson disease, yet as an intrinsically disordered segment it lacks a stable epitope for conventional ligand discovery. Using deep learning-based protein design, we generated compact single-chain binders that take advantage of the propensity of the NAC domain to adopt an extended {beta}-strand conformation, which they engage and stabilise. From 21 expressed designs, 3 engaged the target both in vitro and within live cells. A single round of partial diffusion improved their affinity, maturing the strongest binder to a dissociation constant of 1.94 nM with no detectable cross-reactivity to tau, amylin or amyloid {beta}. Solution NMR spectroscopy confirmed Syn peptide association with all three binders and showed that peptide binding induces conformational changes consistent with the intended design architecture. Each binder engaged a distinct point on the aggregation pathway. Kinetic analysis combining seeded aggregation with the measured affinities for monomer, oligomer and fibril resolved the specific microscopic step that each binder inhibits. Notably, the most potent binder acted by selectively capturing on-pathway oligomers, the species most closely linked to toxicity, and suppressed fibril formation at substoichiometric ratios without engaging the bulk monomer. Together, these findings establish de novo-designed Syn binders that selectively target distinct aggregation intermediates to mechanistically reshape Syn assembly, providing a framework for the rational design of aggregation-modifying proteins.

While lipidation is a widely observed strategy to promote membrane permeation, whether the factors governing lipid-driven accumulation are shared across the divergent membranes of mammalian and Gram-negative cells remains unresolved. Here, we apply the Chloroalkane Azide-based Membrane Penetration (CHAMP) assay to a systematically designed library of lipid conjugates in both HeLa and E. coli cells. CHAMP, developed by our group, pairs a minimally disruptive azide tag with a cytosolically anchored HaloTag to quantify cytosolic accumulation directly. The two systems show divergent trends: most lipid modifications reduce E. coli accumulation, whereas larger, more hydrophobic conjugates, including medium-chain, cyclized, and heteroatom-containing lipids, are preferentially internalized by mammalian cells. Through targeted endogenous and exogenous modifications, we further resolve how charge, scaffold composition, and individual envelope barriers shape these patterns. Together, these results establish that lipidation is a context-dependent permeation principle that fundamentally diverges between mammalian and diderm envelopes. By showing that hydrophobic modifications routinely hinder Gram-negative cytosolic entry, this work explains the scarcity of lipidated Gram-negative antimicrobials, exposes the limits of lipophilicity-driven optimization, and redefines the physicochemical boundaries for penetrating the diderm envelope.

Biomolecular condensates formed through liquid-liquid phase separation (LLPS) compartmentalize biochemical reactions without enclosing membranes, enabling spatiotemporal control over diverse cellular processes. Engineering genetically encoded proteins that phase separate in response to defined chemical inputs remains a central challenge for synthetic biology. Here, we report a coiled-coil peptide polymer, M1, that undergoes cofactor-dependent condensation both in vitro and in Escherichia coli. M1 is an ABA triblock construct comprising two terminal helical domains connected by a flexible, intrinsically disordered linker. The terminal domains are derived from a heme-responsive coiled-coil motif that is destabilized in the apo state but assembles into a four-helix bundle upon metalloporphyrin coordination. We demonstrate that M1 forms condensates exclusively in its cofactor-bound state, both in vitro and in cells. In E. coli, these intracellular condensates accumulate at the cell poles in a concentration-dependent manner. Depletion of cellular heme biosynthetic capacity suppressed condensate formation, which was rescued by supplementation with the heme precursor {delta}-aminolevulinic acid ({delta}-ALA) and iron, consistent with metalloporphyrin coordination triggering assembly. The condensates retain peroxidase activity characteristic of heme-containing proteins and catalyze the oxidation of Amplex Red to resorufin both in vitro and in living cells. These results establish metalloporphyrin binding as a molecular switch for condensate biogenesis in a structured peptide polymer, directly coupling cofactor coordination, mesoscale assembly, and catalytic function within a single designed system. SYNOPSIS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/734084v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@9f7f08org.highwire.dtl.DTLVardef@14cf3dforg.highwire.dtl.DTLVardef@11ee553org.highwire.dtl.DTLVardef@161e109_HPS_FORMAT_FIGEXP M_FIG C_FIG

Human retina can achieve single-photon sensitivity through specialised photoreceptors that convert light into electrical signals via phototransduction. Among microbial light-sensitive proteins, proteorhodopsins stand out for their intrinsic light-driven ion transport and spectral tunability, making them promising candidates for bio-inspired photonic devices. A central challenge for acellular integration, however, is the fragility of most bacterial rhodopsins under extreme conditions. Here, we exploit the exceptional robustness of TARA76, a microbial rhodopsin that retains structural integrity even upon complete dehydration, to demonstrate its functional reconstitution in an artificial black lipid membrane within a biocompatible microfluidic platform. By recording light-induced ionic currents with picoampere sensitivity across a broad range of pH, illumination power, electrolyte composition, and applied voltages, we establish TARA76 as a high-performance photoelectric transducer in a fully acellular environment. Strikingly, we uncover a strong and previously unreported dependence of the photocurrent on Na+ ions, which appears to play a key structural and functional role in stabilising the proteins active conformation. Furthermore, we demonstrate that the orientation of TARA76 within the artificial membrane can be externally controlled by applying a defined electric field during bilayer formation, enabling deterministic tuning of photocurrent directionality. Together, these results establish a robust and miniaturisable bio-photonic platform with direct implications for quantum light sensing, neuromorphic bioelectronics, and next-generation artificial retinal interfaces.

Spns transporters are major facilitator superfamily proteins that regulate lipid transport, lysosomal homeostasis, immunity and disease, yet how protonation enables their chemically diverse transport functions remains unclear. Here, we combine double electron-electron resonance spectroscopy in lipid nanodiscs with DEER- and AlphaFold-guided modeling to define the conformational landscape of the Mycobacterium smegmatis Spns homolog MsSpns. Protonation shifts MsSpns toward an inward-facing state, whereas deprotonation favors a broader outward-facing ensemble through coordinated rearrangements of the intracellular and extracellular gates. These transitions are governed by membrane-embedded protonation switches and proton-sensing networks on both sides of the membrane, while the substrate-binding cavity exhibits distinct proton sensitivity and weaker cooperativity. Hydrophilic cationic substrates, including capreomycin and ethidium bromide, stabilize the outward-facing state, consistent with efflux antiport, whereas lipophilic compounds, including rifampicin, epicholesterol and certain phospholipids, favor the inward-facing state, suggesting uptake or allosteric stabilization. Thus, conserved proton-coupling elements can drive substrate transport in opposite directions, revealing the mechanistic versatility of the Spns fold with therapeutic potential.

Phosphorylation of intrinsically disordered proteins (IDPs) is essential for regulating biomolecular interactions in many cellular processes. However, a quantitative understanding of how phosphorylation tunes the affinity between highly charged IDPs and nucleic acids is lacking. Here, we show that multi-site phosphorylation of the disordered arginine/serine-rich (RS) domain of the splicing factor SRSF1 acts as an electrostatic rheostat that governs RNA binding. By combining enzymatic phosphorylation, phosphomimetic variants, and chemically synthesised phosphopeptides with single-molecule Forster resonance energy transfer measurements, we reveal the RS domain to be a potent driver of protein-RNA association. Increasing phosphorylation progressively reduces this interaction, and extensive phosphorylation eliminates detectable RNA binding. Remarkably, the binding free energy depends linearly on RS-domain net charge, regardless of whether the charge arises from phosphorylation or acidic residues introduced as phosphomimetics. Together, our findings uncover a quantitative framework for how phosphorylation tunes the interactions of charged IDPs and rationalize why two acidic residues are required to mimic a single phosphorylation event.

Stereochemistry plays a crucial role in how molecules interact with complex physiological environments, affecting pharmacokinetics, pharmacodynamics, efficacy, and toxicity. Although these effects are well studied for small-molecular drugs, they are largely overlooked for supramolecular assemblies used in drug delivery. Even for lipid nanoparticles (LNPs)--the most advanced RNA delivery platform--stereochemical effects are rarely investigated and, when considered, are typically limited to the ionizable lipid rather than the overall stereochemical identity of the LNP. Here we separate the ionizable lipid cKK-E12 into its two stereoisomers (trans: R,S/S,R; cis: R,R/S,S), which are normally used as a mixture. LNPs containing the cis isomer exhibit improved physicochemical properties, stability, and protein expression. By systematically varying the stereochemistry of the ionizable lipid, phospholipid, and cholesterol, we reveal stereochemistry-dependent differences in uptake and protein expression across six cell lines and in vivo in zebrafish embryos and mice. AI-assisted cryo-TEM analysis and SAXS link enhanced protein expression to structural differences, demonstrating control over internal lipid phases (lamellar and inverse hexagonal), influencing sample uniformity, and identifying stereochemical identity as a key determinant of functional RNA delivery.

Monoterpenes are a diverse class of natural products with broad industrial and pharmaceutical applications. While there is great interest in transitioning their production from chemical synthesis and natural source extraction to yeast bioprocesses, this approach remains limited by the dual functionality of the endogenous farnesyl diphosphate synthase Erg20p, which produces the monoterpene precursor geranyl diphosphate (GPP) but favors its subsequent conversion to farnesyl diphosphate (FPP). To address this limitation, we recruited Erg20p and monoterpene synthases into synthetic membraneless organelles, improving production. In doing so, we found that short C-terminal peptide fusions used for recruitment also significantly enhance GPP synthase activity relative to FPP synthase activity. The combined effects of metabolic spatial organization and GPP synthase activity enhancement significantly boost production of different monoterpenes, including geraniol titers exceeding 4 g/L. The strategies presented here can be readily integrated with other traditional metabolic engineering approaches to build yeast strains with high levels of monoterpene production.

Directed evolution has long been constrained by complex screening hardware and labor-intensive workflows. Here, we report the first genuine test-tube screening platform that uses His6-tagged peptide-functionalized magnetic beads and Fe3+-decorated E. coli cells to establish a phenotype-genotype linkage, thereby decoupling ultrahigh-throughput screening from specialized instrumentation and democratizing directed evolution. The platform demonstrated a screening throughput of > 108 events s-1 and an enrichment factor of up to 63-fold. Using galactose oxidase as a model, we identified variants with up to a 26-fold increase in catalytic efficiency. Extensions to D-amino acid oxidase and alcohol oxidase yielded variants with up to 5383-fold and 25-fold improvements over their respective wildtypes after a single round of screening. These results highlight the platforms capacity to rapidly engineer H2O2-generating oxidases and to advance AI-driven enzyme design through rapid data generation.

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

-Synuclein (S), a protein that plays a central role in Parkinsons disease and related synucleinopathies, is an intrinsically disordered protein (IDP) whose functional interactions and aggregation behavior can be strongly influenced by post-translational modifications (PTMs). Phosphorylation, acetylation, and other PTMs regulate Ss interactions with lipid membranes and binding partners, whereas their dysregulation is associated with aggregation and neuronal toxicity. Despite significant progress through chemical and semi-synthetic approaches, investigating the combinatorial effects of PTMs has remained challenging due to the lack of accessible, site-specific methods. Here, we present an integrated strategy combining genetic code expansion, enzymatic modification, and intein-mediated click chemistry to generate S variants bearing multiple defined PTMs and a C-terminal fluorescent label. The resulting constructs enable direct evaluation of how individual and combined PTMs influence S structure, lipid binding, and cellular internalization. Our approach expands the molecular toolkit for dissecting PTM crosstalk in S and other aggregation-prone IDPs, advancing mechanistic understanding and supporting the development of therapeutic strategies for neurodegenerative disease.

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

Phase separation (PS) of the low-complexity domain (LCD) of TDP-43 is linked to pathogenic aggregates in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). Here, we show that extensive phosphorylation of the LCD C-terminus redirects its self-assembly. Coarse-grained Monte Carlo simulations predicted that 12 Ser phosphorylations partition the 148-residue LCD into a hydrophobic N-terminal and highly charged C-terminal block, favoring finite-sized micellization over macroscopic PS. In vitro, LCD phosphorylated by casein kinase 1 delta (CK1{delta}; mean of 12 phosphorylations by native mass spectrometry) and phosphomimetic 12D/12DD mutants formed spherical nanoparticles ({approx} 20-50 nm) above a low-micromolar critical micelle concentration, whereas the unphosphorylated LCD underwent reversible PS that matured into fibrils. Increasing ionic strength shifted the mutants toward anisotropic morphologies (worm-like 12D micelles and rigid 12DD nanocylinders). Turbidity assays and confocal imaging directly visualized the absence of PS in the phosphorylated form. Negative-stain and cryo-EM confirmed the spherical micellar architecture for the phosphorylated LCD and 12D/12DD mimics. Our data identify phosphorylation as a molecular switch tuning macrophase separation and fibril formation of TDP-43 LCD, providing a framework for an aggregation-protective role through microphase separation into size-limited micelles. Whether these assemblies are stable or kinetically trapped on pathological timescales remains unclear.

Amyloid fibrils are intrinsically polymorphic protein assemblies that form distinct structural strains linked to diverse biological and pathological outcomes. Yet, the principles governing how sequence encodes diverse fibril architectures, and the extent to which a given fold constrains underlying amino-acid sequence compatibility, remain poorly understood. Here, we apply generative protein design to directly interrogate the sequence-structure relationship of defined fibril architectures, using -synuclein (S), a protein known to form highly polymorphic amyloid fibrils, as a model system. Sampling sequence space under structural constraints reveals a continuous compatibility manifold in which diverse sequences encode a common amyloid architecture. De novo designed sequences assemble into fibrils, often with enhanced aggregation efficiency relative to S. A subset exhibits strain-like behaviour, including similar morphologies, efficient cross-templating, and induction of S cellular propagation, thereby functionally validating structural compatibility with the native fibril fold. Energetic analysis shows that stability is achieved through distinct but compensatory interactions, supporting a non-unique mapping between sequence and structure. Together, our results define a continuous and constrained compatibility landscape underlying amyloid strains, providing a framework for understanding the determinants of polymorphism and establishing generative protein design as a strategy to access this space, interrogate amyloid sequence-structure relationships, and engineer fibrillar protein assemblies and functional biomaterials.