Nature Chemistry

Early ribonucleoprotein systems likely required mechanisms to concentrate and organize RNA. Modern ribosomes use Mg2+ and evolved Lys/Arg-rich protein extensions to stabilize their RNA backbone. Before templated synthesis, however, peptide formation likely generated heterogeneous sequence populations rather than reproducible ribosomal sequences. Here, we compare Mg2+, ribosomal peptides, and statistical peptide libraries in organizing a 136-nucleotide model of the peptidyl transferase center (sPTC). Mg2+ condenses sPTC only at a large excess of positive charge following thermal annealing, producing largely arrested libraries. Acidic conditions further promote condensation. Ribosomal peptides instead promote coacervation near charge stoichiometry and form droplets that readily fuse. Several statistical peptide libraries also coacervate with sPTC, despite comprising heterogenous mixtures rather than a single defined sequence. Increasing mean positive charge favors condensation, but Lys/Arg-containing libraries undergo liquid-liquid phase separation more readily and across broader conditions than matched libraries containing the prebiotically plausible diaminopropionic acid (Dpr) and diaminobutyric acid (Dab). Atomistic computer simulations implicate Arg as a major source of this difference, as it can form more numerous and longer-lived hydrogen bonds with RNA, while competitive partitioning experiments show preferential condensate recruitment in the order Arg > Lys > Dab > Dpr. Together, our findings show that peptide-RNA coacervation can be triggered collectively by statistical peptide ensembles, providing a plausible route to protoribosomal organization without peptide sequence-specific optimization. Cationic chemistry shapes condensate formation and material properties, suggesting that amino acid alphabet formation could have broadened the conditions supporting liquid-like protoribosomal assemblies.

The ability to map protein interactomes and organelle proteomes is foundational for achieving a molecular understanding of living cells. Proximity labeling (PL) provides a powerful strategy for this, but existing enzymes and photocatalysts are limited by their spatial resolution, reliance on biotin, and/or in vivo compatibility. Here we report FlexID, an engineered promiscuous ligase that catalyzes the rapid attachment of diverse small-molecule probes to proximal endogenous proteins. Critically, FlexID operates through a zero-radius, direct-contact mechanism, offering superior spatial precision compared to existing PL tools. We engineered FlexID by combining the strengths of sequence- and structure-trained computational models to enhance its catalytic activity and structural stability. Biophysical analysis revealed that specific conformational changes in FlexID improve its ability to recognize diverse target proteins while simultaneously preventing the premature release of the reactive intermediate. We demonstrate FlexID's versatility through in vivo proximity labeling, comprehensive organelle proteome mapping, and a high-throughput, fluorescence-based screen for molecular glues. Our work shows that computational methods can be harnessed to create mechanistically distinct PL enzymes and establishes FlexID as a flexible, high-resolution tool for mapping protein interactions and proteomes in living cells.

Catalyst-controlled stereodivergent synthesis of acyclic molecules bearing multiple stereogenic centers remains a longstanding challenge in asymmetric catalysis, particularly through intermolecular radical C-C coupling. Here, we report the directed evolution of pyridoxal 5'-phosphate (PLP)-dependent biocatalysts for the stereodivergent synthesis of non canonical amino acids bearing either skipped , {gamma}-stereochemical dyads or contiguous , {beta}, {gamma}-stereochemical triads. Starting from a thermophilic tryptophan synthase {beta}-subunit, protein engineering generated complementary enzyme variants that selectively construct three of the four possible , {gamma}-stereoisomers through biocatalyst-controlled reversal of both remote {gamma}-stereochemistry and proximal -stereochemistry. These engineered enzymes catalyzed intermolecular radical C-C bond formation with a broad range of secondary alkyltrifluoroborates, affording , {gamma}-disubstituted amino acids in excellent yields and stereoselectivities. Furthermore, directed evolution enabled efficient {beta}-dehydroxylative radical coupling of the non-native threonine-derived aminoacrylate intermediate, providing amino acids bearing contiguous , {beta}, {gamma}-stereochemical triads with up to 96:4:<0.1:<0.1 d.r. and >99:1 e.r.. To further demonstrate the synthetic utility of this platform, the stereochemically enriched amino acids were transformed into valuable {gamma}-chiral amines through engineered PLP dependent tryptophan decarboxylases, establishing a dual PLP enzyme based biocatalytic cascade for the enantiodivergent synthesis of privileged amine scaffolds. Molecular dynamics simulations reveal that enzyme controlled {gamma}-stereoselectivity originates from active-site remodeling that modulates {pi}-{pi} interactions and steric contacts governing the facial selectivity of the radical addition step. This work expands the synthetic capabilities of radical biocatalysis by enabling catalyst-controlled stereodivergent assembly of complex acyclic stereochemical arrays and establishes engineered PLP enzymes as versatile catalysts for the synthesis of stereochemically complex amino acids and chiral amines.

Biomolecular condensation emerges from networks of multivalent interactions that respond collectively to molecular composition and environmental conditions. Here, we establish a sequential Design of Experiments (DoE) framework to systematically and efficiently characterise multidimensional condensation landscapes. Applied to three RNA-binding condensate proteins - Dhh1, Ded1 and PGL-3 - the workflow revealed unexpected protein-specific interactions between biophysical and biochemical variables, including RNA, pH and ionic strength, and uncovered context-dependent switch-like behaviours. Extending the framework to protein mixtures revealed how proteins affected each other's condensation and whether they formed condensate-within-condensate structures under different experimental conditions. Finally, an unexpected response identified by DoE prompted mechanistic analysis of Dhh1, revealing an ATP-independent RNA-binding surface that promotes efficient condensation in vitro and contributes to P-body formation in cells. Sequential DoE therefore provides an experimentally accessible strategy for uncovering context-dependent condensate regulation and converting multidimensional response patterns into mechanistic hypotheses.

The fosfazinomycins are phosphonate natural products with antifungal activity. Their main scaffold is composed of a phosphonate moiety attached to the carboxylate of arginine via a hydrazine linkage. Previous studies have elucidated a convergent biosynthetic pathway that independently assembles phosphonate and hydrazine synthons, but how these building blocks are then connected could not be determined. In this work, we provide the final missing steps in fosfazinomycin biosynthesis by revealing an unexpected engagement of biosynthetic machinery that is typically involved in ribosomally synthesized and post-translationally modified peptides (RiPPs). An asparagine synthetase-like (AS-like) enzyme catalyzes the installation of hydrazine onto the carboxylate of Arg at the C-terminus of a short ribosomally synthesized precursor peptide. The terminal nitrogen of the resulting peptide hydrazide is methylated, and the phosphonate moiety is activated to a triphosphate-like intermediate through two separate kinase catalyzed phosphorylation steps. A nucleotidyl transferase then catalyzes the ligation of the two fragments to generate the mature fosfazinomycin scaffold on a peptide. Aminopeptidase cleavage of this peptide then yields fosfazinomycin B (fosB), which serves as substrate for a valinyl-tRNA dependent reaction to afford fosfazinomycin A (fosA). This work demonstrates an unprecedented example of the convergence of RiPP and phosphonate biosynthetic logic, an enzymatic route to peptide hydrazides that are widely used in peptide ligation chemistry, and an unusual activation sequence for conjugation of phosphonates.

Precise organization of bioactive ligands at material-cell interfaces is critical for directing integrin-mediated cell behavior, yet tuning supramolecular organization commonly necessitates orchestrated redesign of molecular composition or ligand sequence. Here we establish terminal modification as a minimal, sequence-preserving strategy to reprogram the organization and function of self-assembling integrin ligands. Using FFFIKVAV as a model system, N- and C-terminal modifications generated distinct supramolecular states with markedly different integrin activation and cell migration responses. Notably, stronger intermolecular association did not produce stronger biological activity, as N-terminal acetylation promoted scaffold consolidation while reducing the configurational freedom of the exposed IKVAV ligand and diminishing integrin activation and cell migration. Alongside modulating migration magnitude, terminal chemistry also diversified the cellular programs supporting migration, with the parent peptide and its C-terminally amidated analogue favoring protrusion-dominant and stress-fiber-dominant adhesion-cytoskeletal programs, respectively. These functional states were reproduced when the assemblies were introduced directly to pre-adherent cells, extending their activity beyond preformed material coatings. Our findings identify terminal chemistry as a compact molecular design variable that links supramolecular organization to ligand function while diversifying the biological output of a shared bioactive sequence.

Directed evolution and machine-learning-assisted evolution have transformed biocatalysis by enabling rapid optimisation of catalytic performance within compatible protein scaffolds. In parallel, advances in deep-learning-driven de novo protein design have expanded accessible protein architectural space, enabling the generation of functional catalysts for increasingly challenging transformations. We herein report a synergistic design-evolution strategy to encode new-to-nature regio- and enantioselective C-H amination reactions using engineered de novo haemproteins to synthesise chiral piperidines, a privileged pharmacophore in drug discovery. By integrating motif scaffolding, computational re-design, and directed evolution, we demonstrate de novo enzyme design can enable challenging, high-energy-barrier enzyme chemistry with programmable regio- and enantioselectivity, high catalytic efficiency, excellent enzyme stability, and scaffold diversity.

Despite their intriguing native metalloenzymology, naturally occurring copper enzymes remain largely underexploited for new-to-nature biocatalytic reactions. Herein, we report the systematic investigation and reprogramming of natural copper enzymes to catalyze unnatural free radical (pseudo)halogenation reaction in a highly enantioselective fashion. Evaluating Cu enzymes in the decarboxylative azidation of redox-active esters revealed activity across multiple Cu enzyme families, with type III binuclear Cu enzymes, particularly the Bacillus megaterium tyrosinase (BmTyr), exhibiting superior activity and enantioselectivity across both stabilized and unstabilized alkyl radicals upon further engineering. The strong halide binding affinity of the binuclear Cu system also enabled challenging enantioconvergent bromination, chlorination and isothiocyanation reactions, which remained inaccessible to repurposed nonheme Fe enzymes. Further EPR and UV-visible spectroscopic analyses confirmed the coupled binuclear nature of wild-type and engineered bacterial tyrosinases and provided insights into the origin of their enhanced activity. Collectively, this study establishes binuclear copper enzymes as a powerful platform for new-to-nature stereoselective radical reactions, expanding the scope of metalloenzyme catalysis beyond mononuclear systems.

Fluorescent proteins (FPs) are the workhorse of modern-day cellular imaging and biosensing. However, rationally predicting and engineering the photophysical properties of FPs remain challenging, as these are governed by complex three-dimensional microenvironments and post-translational modifications. Here, we present a novel surface-mutation-guided engineering strategy combined with iterative rounds of rational mutagenesis to improve key photophysical properties of red fluorescent proteins (RFPs). This approach generated two bright, monomeric RFP variants from the mFruits lineage, mCranberry and mCranberry2, with quantum yields of 85% and 80%, respectively. Strategic incorporation of charged amino acid residues on the protein surface enhanced fluorescence quantum yield without compromising photostability. Comprehensive photophysical characterization and live-cell microscopy demonstrate enhanced performance of these variants and establish their utility as robust probes for fluorescence imaging. However, the exact mechanism of such improvements is subject to further investigation. Collectively, this work introduces a strategy for optimizing fluorescence properties and provides a framework that may be broadly applicable to other pigment-protein systems such as genetically encoded metal-ion indicators and photosynthetic light-harvesting complexes.

Despite rapid advances in new-to-nature biocatalysis, trimolecular enzymatic reactions that generate previously inaccessible molecular entities, particularly those bearing well-defined, congested stereochemical dyads, remain exceedingly rare. Here, we report a novel diastereo- and enantioselective photobiocatalytic three-component radical coupling unknown in both organic chemistry and enzymology, enabled by cooperative catalysis employing an evolved pyridoxal biocatalyst and a transition-metal photosensitizer. Directed evolution using high-throughput photobiocatalysis furnished two threonine aldolase variants, enabling the stereoselective assembly of contiguous tri- and tetrasubstituted stereocenters as well as vicinal tetrasubstituted stereocenters, which were long regarded as a challenging goal for stereoselective radical chemistry and asymmetric catalysis. Furthermore, a variety of radical precursors, including alpha-iodoesters, alpha-iodoamides, alpha-iodoketones, and alpha-iodonitriles, could be transformed into structurally diverse, densely substituted non-canonical amino acid derivatives via C-H functionalization with excellent stereocontrol. Leveraging the broad substrate compatibility of engineered pyridoxal biocatalysts, combinatorial biocatalysis was achieved by simultaneously varying all three coupling partners, delivering products with a 98% success rate. Collectively, this diversity-generating multicomponent photobiocatalytic radical coupling afforded a powerful strategy for addressing long-standing challenges in asymmetric catalysis while enabling access to valuable stereochemically complex small molecules with applications in medicinal chemistry.

Designing photochemical properties in proteins is a frontier challenge in biomolecular design, which, when realised, will open new avenues in catalysis and biomolecular electronics. Here we demonstrate an integrated computational design framework, supported by ultrafast spectroscopy, combining protein design, quantum mechanical/molecular mechanics molecular dynamics (QM/MM MD) and electronic structure calculations, to control flavin excited states in de novo proteins. Iterative design yields FC-4D2, a flavocytochrome with covalently bound flavin and non-covalently bound heme B and Fl-4D2, where the heme-binding site is removed. Both proteins are highly thermostable, and access all physiologically relevant flavin redox and protonation states, while high-resolution X-ray crystallography confirms the near-[A]ngstrom-level accuracy in design. Incorporating QM/MM MD and linear-response time-dependent density functional theory (LR-TDDFT) into the design workflow enables engineering of a flavin-tryptophan charge-transfer variant, Fl-4D2CT, that modifies the flavin absorption spectrum, substantially alters its redox properties and suppresses fluorescence. Ultrafast studies reveal how protein design reshapes the excited state relaxation timescale and mechanism. Our study establishes a modular strategy for programming flavin photochemistry in designed proteins.

The prevailing view holds that simpler animate beings predating the dawn of cellular life once existed, yet they have long since vanished from the present-day Earth. Our hypothesis posits that these animate beings persist to this day. To this end, we devised a long-term experiment spanning over a decade, keeping Staphylococcus aureus (S. aureus) within a sealed aqueous system of pure water. The findings illustrated that, upon the complete cell death (CCD) of the S. aureus, as cellular remnants progressively succumbed to dissolution, the aqueous solution underwent self-purification, bringing forth a class of lipid bilayer membrane microspheres containing RNA and proteins. These structures maintained long-term sustainable stability within the thermodynamic storm of water molecules and underwent dynamic size fluctuations over time - collectively exhibited phenomena indicative of being "alive". This study reveals that the cell, upon complete cell death, undergoes reversion to a living entity simpler than the cell itself - the Meta Body (MB). The discovery of MB opens an entire re-conceptualization of what death ultimately brings.

Abstract Peptide lasso-grafting strategies enable the transfer of established pharmacophores into compact protein scaffolds but often require optimization to preserve target binding and biological activity. Here, we report Random in-vitro Protein single-Loop Engineering (RiPLE), an mRNA-display strategy for the direct discovery of engineered ubiquitin binders, termed U-bodies, from a library containing a fully randomized surface loop of variable length. Using receptor tyrosine kinase-like orphan receptor 1 (ROR1) as a model target, we generated U-bodies through RiPLE and compared them with lasso-grafted U-bodies derived from a previously identified ROR1-binding macrocyclic peptide. Both approaches yielded U-bodies bearing single-digit nanomolar affinity to ROR1; however, RiPLE converged on de novo binding sequences that were distinct from the parental peptide. Notably, most selected U-bodies exhibited strong selectivity for ROR1 over the closely related receptor ROR2, whereas the parental peptide bound both receptors with high affinity. Representative ROR1-binding U-bodies also inhibited wound closure in ROR1-expressing MDA-MB-231 cells without detectable cytotoxicity. Applying RiPLE to the heterotrimeric G-protein subunit (GS) similarly yielded high-affinity binders and revealed sequence convergence patterns distinct from lasso-grafting. Together, these results establish RiPLE as a powerful and complementary alternative to peptide-guided lasso-grafting, enabling de novo evolution of functional binding surfaces directly within their final protein scaffold.

In retinal proteins, light absorption leads to a double bond isomerization of the chromophore, followed by conformational relaxations that drive functional protein rearrangements. The balance between photoisomerization and radiative or non-radiative decays of the excited state is adjusted to the demands of the respective photoreceptor function. The molecular determinants controlling the excited state processes remain poorly understood and are the focus of this combined crystallographic, spectroscopic, and theoretical work on the recently discovered neorhodopsin (NeoR). NeoR exhibits a highly red-shifted and fluorescent dark state and selectively photoisomerizes from all-trans to 7-cis. We show that the local electrostatic environment reduces the negative charge density near the Schiff base and suppresses torsional motion around the canonical C13=C14 isomerization site of microbial rhodopsins. Instead, the chromophore-binding pocket permits only low-efficiency C7=C8 isomerization, accompanied by polyene inversion, {beta}-ionone ring displacement, and Schiff base deprotonation. Consequently, unlike other retinal photoreceptors, NeoR undergoes structural changes localized near the {beta}-ionone ring.

Self-labeling protein (SLP) tags enable versatile labeling of proteins in live cells, yet only two SLPs are commonly used, limiting multiplexing. Here, we repurposed the oncoprotein K-Ras(G12C) and its covalent inhibitors to create a third, orthogonal SLP system. We used a deep-learning-based approach to radically redesign the sequence of K-Ras(G12C) while preserving its covalent-inhibitor binding pocket. Our top design, LUCI-tag, is a 19 kDa, monomeric, thermostable SLP that rapidly and covalently reacts with commercially available K-Ras(G12C) inhibitors bearing diverse payloads. Unlike existing SLPs, LUCI-tag exhibited payload-agnostic labeling kinetics, outperforming HaloTag7 and SNAP-tag for a negatively charged payload. X-ray crystal structures of drug-bound and drug-free LUCI-tag showed the design was structurally accurate and contained a preorganized inhibitor-binding pocket that could explain its rapid labeling kinetics. Proteomics and cell-signaling experiments confirmed LUCI-tag is biologically inert. LUCI-tag enabled rapid, wash-free, live-cell imaging and simultaneous three-color multiplexed experiments with HaloTag7 and SNAP-tag. This work establishes LUCI-tag as an immediately useful orthogonal SLP and demonstrates that covalent drug-target pairs can be repurposed into a broadly applicable platform for protein labeling.

Methotrexate (MTX) is a narrow-therapeutic-index drug widely used in cancer and autoimmune disease treatment, for which delayed clearance can cause severe toxicity and requires close therapeutic drug monitoring. Here, we integrate high-stringency Capture-SELEX, biophysical characterization, molecular modeling, mutational validation, and electrochemical transduction to develop two structurally distinct MTX-binding DNA aptamer families. Full-length 3315 and 930 bound MTX with apparent KD values of 89 and 408 nM, respectively. Systematic truncation generated compact functional variants: 3315-55 retained 63 nM affinity and broad buffer tolerance, whereas 930-65 reached an apparent KD of 44 nM in 10 mM Mg2, revealing markedly different ion dependencies. Biolayer interferometry confirmed rapid and reversible MTX recognition, while 3315 variants retained binding in serum-containing buffer. Computational analysis and site-directed mutagenesis identified distinct recognition architectures: a compact G-rich scaffold coupled to a central binding interface in 3315 and a localized Mg2+-stabilized binding pocket in 930. Methylene blue-labeled 3315-55 and 930-65 were integrated into electrochemical aptamer-based sensors, producing concentration-dependent responses to 100-1000 uM MTX in PBS, discriminating MTX from folic acid, and responding to MTX directly in undiluted human blood. Together, these results establish a structure-guided workflow linking aptamer discovery, mechanistic validation, and electrochemical translation, providing new molecular recognition elements and a foundation for future reagent-less MTX therapeutic drug monitoring.

Cytokines can elicit potent antitumor immunity, but their clinical use is often limited by systemic immune activation and dose-limiting toxicity. Protease-responsive masking offers a way to improve selectivity by linking cytokine activity to disease-associated protease cleavage, but current approaches have faced several challenges. Here we describe a de novo protein design strategy for generating protease-activatable cytokine prodrugs that overcomes these challenges. We use this strategy to design and characterize cytokine prodrugs and well-behaved AND-gated split systems that require both target-dependent colocalization and proteolytic unmasking to generate active cytokines. In challenging syngeneic tumor models, our IL-21 prodrugs reduced treatment-associated toxicity while maintaining antitumor efficacy.

Physiological compartments span a wide pH range, from blood plasma near pH 7.4 through the mildly acidic tumor microenvironment to the acidified endosome and lysosome, and binders whose affinity responds to these differences underpin recycling antibodies, catalytic extracellular degraders and tumor-conditional therapeutics. Such binders are still obtained mainly by combinatorial histidine scanning and display selection, which is laborious and yields few transferable design rules. Recent computational approaches have enabled the design of pH-sensitive binders that engage at physiological pH and release under acidic conditions, by introducing histidines at key positions so that protonation generates electrostatic repulsion, evaluated through physics-based calculations. However, because the design model has no explicit representation of protonation state, it cannot shape the residues around a titratable site to favor one state over the other, leaving pH sensitivity to be screened for rather than specified. Here we present Proton-PottsMPNN, a sequence-design model that explicitly represents the protonated and deprotonated states of histidine, aspartate and glutamate as distinct sequence tokens. Because protonation state is specified rather than inferred, the model redesigns the microenvironment around a titratable residue so that the chosen protonation state is energetically favored over its alternative. Using Proton-PottsMPNN we designed a library of 8,407 de novo PD-L1 candidate binders in which sequence-structure compatibility and protonation state preference were designed jointly under a single energy function, with no structure-prediction filtering applied. The full library was synthesized and displayed on yeast, then screened against PD-L1 across a series of pH values by fluorescence- and magnetic-activated cell sorting, recovering 237 and 288 unique pH-dependent designs respectively. Transition pH values spanned 4.0 to 5.8, and switches were centered on protonated aspartate and glutamate as well as histidine. Recovered designs showed a stronger model preference for their designed protonation state than designs from the same backbone that were not recovered, and this preference was strongest in the designs that required the most acidic conditions to bind. Proton-PottsMPNN therefore designs binders that engage PD-L1 under acidic conditions fully de novo, directly from a specified protonation state.

Generative protein design has made sequence and structure programmable, yet its chemical alphabet remains largely confined to the canonical twenty. Here, we establish CheMoDesign, an experimentally validated generative framework for designing proteins de novo around user-specified chemistry. CheMoDesign weakens pair-representation conditioning in a pretrained all-atom structure-prediction model while preserving chemical anchors, then refines generated backbones through sequence-conditioned denoising. By treating post-translational modifications, synthetic residues, and chemical modifications as user-defined building blocks, the framework generated proteins with chemistry-dependent recognition and reactivity in single-round experimental campaigns. Sulfotyrosine and nitrotyrosine enabled modification-dependent recognition of thrombin and HER2, respectively. Genetically encoded noncanonical residues enabled bioorthogonal affinity switching and light- or proximity-triggered covalent target capture. Post-expression chemical modifications yielded a light-activated covalent PD-L1 binder and an isoform-selective CA IX inhibitor (KD = 45 nM) designed to engage catalytic Zn2+;. CheMoDesign makes chemical mechanism a programmable dimension of de novo protein design.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are produced from precursor peptides that typically contain a short, unstructured N-terminal leader region that engages RiPP biosynthetic enzymes through peptide-protein contacts. A distinct subclass of leader peptides, the nitrogen-fixing protein 11-like leader peptides (Nif11-LPs), are unusually long and conspicuously enriched in cyanobacteria. No atomic structures of Nif11-LPs have been reported. Here, we report the solution structure of MppEleader, a Nif11-LP from the marine cyanobacterium Moorena producens PAL. MppEleader adopts a bipartite architecture with a rigid N-terminal tetrahelical bundle stabilized around a hydrophobic nucleus, followed by a dynamic, largely disordered C-terminal region containing a conserved L(X)L motif. We show that the -ketoglutarate/non-heme iron enzyme MppG dihydroxylates the macrocyclized MppEcore peptide in a strictly leader-dependent manner. NMR titrations and structure-guided mutagenesis demonstrate that both the tetrahelical hydrophobic nucleus and the L(X)L motif of MppE make direct, functionally required contacts with MppG, including its RiPP recognition element (RRE) domain. An AlphaFold3-guided structural model rationalizes these contacts. Together, these findings establish a tertiary structure-dependent protein-protein interaction paradigm for RiPP core peptide modification, distinguishing Nif11-LP recognition from canonical short-leader mechanisms and expanding the understanding of substrate recognition in RiPP biosynthesis.