Nature Chemistry

Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins underlies the formation of biomolecular condensates that regulate diverse cellular processes, while its dysregulation contributes to protein aggregation and disease. Despite its importance, molecularly defined and target-specific strategies to control LLPS remain limited. Here, we present a systematic framework for designing de novo peptides that induce and modulate LLPS of -synuclein. By integrating deep mutational scanning with peptide screening, we identified sequence features that govern condensate formation and enabled the creation of optimized peptides with high efficiency and specificity. Biophysical analyses revealed that LLPS efficiency is dictated by the interplay of solubility, multivalency, and cooperative interactions, resulting in a distinctive bell-shaped phase diagram. Thermodynamic measurements and imaging-based analyses further demonstrated that condensate stability and material properties can be rationally tuned through peptide optimization. Together, these findings establish generalizable design principles for engineering LLPS modulators in biologically and pathologically relevant protein systems.

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.

Pathological seeding of protein misfolding is a hallmark of proteinopathies. However therapeutic strategies to clear these aggregates are lacking, impairing both study of their biological importance in disease etiology and progression as well as development of therapeutics. This is due in part to the need to selectively clear oligomerized proteins whilst leaving functional monomers intact, as well as the challenge of developing molecules that act on the full complement of misfolds the protein can adopt throughout the course of disease. In this work, we describe a dopant system consisting of an engineered alpha-synuclein protein construct that rapidly co-aggregates into existing WT alpha-synuclein oligomers, enabling rapid degradation of the entire assembly in the presence of a small molecule trigger. This work provides proof-of-principle for an approach that transforms pathological seeding from a disease-driver into a therapeutic vulnerability, and is potentially applicable to any proteinopathy without requiring a small molecule binder of the pathologic species.

Understanding the effects of formulation excipients on protein solubility is a key part of physical chemistry and pharmaceutical sciences. While excipients are routinely employed to reduce the self-association of biologic drugs, their mechanisms of action remain poorly understood and are often assumed to be broadly nonspecific. Using a high-throughput combinatorial droplet microfluidic platform, we systematically survey and quantify how common pharmaceutical excipients affect the solubility of a diverse panel of therapeutic monoclonal antibodies (mAbs). We show that, while excipients are generally solubilizing, their effects vary substantially across different mAbs, with excipient-specific solubilization scores spanning dynamic ranges of approximately 7-fold to >200-fold across the antibody panel. Histidine, arginine and sodium chloride, in particular, engage in interactions characterized by unique molecular specificity, whereas sucrose effects are largely governed by nonspecific, solvent-mediated interactions. Correlating excipient performance with dynamical mAb molecular features from solvated full-length homology models allows us to dissect and quantify the relative contributions of molecular features governing excipient-mediated solubilization. We envision this new physicochemical understanding lays the groundwork for rational excipient selection and bespoke formulation design, with direct implications for accelerating protein therapeutic development for preclinical scenarios.

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.

RNA protection and controlled release are critical for both fundamental research and therapeutic applications, yet the development of simple, efficient, and reversible post-synthetic RNA modification strategies remains a significant challenge. Here, we introduce a straight forward approach based on ribose 2'-hydroxyl acylation with a nitro-functionalized carbamate, which acts as a redox-responsive center. This modification selectively inhibits native RNA function while remaining inert to endogenous biogenic reductants and common reducing agents used in biological assays. Upon treatment with low millimolar concentrations of the THDB- BIPY reducing pair, RNA function is rapidly restored on a minute timescale. This methodology is broadly applicable across diverse RNA classes and functional contexts, including synthetic RNA oligomers, fluorogenic RNA aptamers, single-guide RNAs in CRISPR-Cas9 gene-editing systems, and mRNAs in living cells for translation control and RNAi-mediated gene silencing. These results demonstrate the general utility of this approach as a chemically controllable functional switch, providing a versatile toolkit for temporal regulation of RNA activity in both research and biotechnological applications.

In their recent communication in Angewandte Chemie (10.1002/anie.202508565), Suk Min Kim and coworkers have described the effect of modifying the gas channels of the CO dehydrogenase II from Carboxydothermus hydrogenoformans, an enzyme that oxidizes reversibly CO into CO2. Their goal was to use mutagenesis to slow down the arrival of O2 at the active site. They reported a large increase in the resistance against oxygen, one of the major barriers to the application of this extremely fast and efficient enzyme in biotechnological devices, with an increase in the IC50 of more than two orders of magnitudes for some variants, with only a minor impact on the affinity of the enzyme for CO. We have produced the same variants, and characterized them in depth using Protein Film Electrochemistry. We used an approach that has proven very useful to learn and understand about the reactivity of CO dehydrogenases (and other redox enzymes like hydrogenases) with O2. We found that, contrary to the claims by Kim and coworkers, the A559W and the A559W/V610H mutants are not more resistant than the WT against oxygen.

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.

Nucleic acid manipulation using programmable ribonucleoprotein complexes (RNPs) has enabled transformative research tools and led to new therapeutic strategies. RNA directly regulates diverse cellular processes,1 is a crucial mediator of protein synthesis, and offers advantages in therapeutic targeting and fundamental discovery complementary to those of DNA.2 ISC-3 and Cas-based4,5 scaffolds where the RNP is fused to an effector protein can alter RNA sequence, structure, and function. However, the non-human origins underlying these systems create challenges in therapeutic translation and the presence of non-native proteins can have unintended and little understood effects on cells.6-8 Systematic repurposing of human proteins, which have been optimized in the cellular environment by evolution, for expanded programmable functions could reveal new biological principles and bypass the limitations of foreign proteins. Here, we demonstrate that the catalytic engine of the RNA-interference (RNAi) pathway, human Argonaute 2 (AGO2),9,10 can be repurposed as a modular targeting domain, and when fused to a C-to-U deaminase, enable AGO-Led Targeted Editing of RNA (ALTER). Using guide RNAs which remodel target RNA structure for selective editing and reduced nuclease activity, we show that ALTER can act on a variety of target transcripts including endogenous mRNAs and lncRNAs, with activities comparable or exceeding those of Cas-based systems.11,12 Despite its human origin and role in RNAi, transcriptome-wide RNAseq revealed lower levels of off-target editing compared to Cas-based editing systems. These results demonstrate that AGO2 can be rationally redirected from RNAi to a broader spectrum of RNA manipulations, establishing that intact human proteins can be reconfigured for expanded molecular function.

Ionic liquids (ILs) are designer solvents with tunable properties that have emerged as powerful cosolutes in protein science and biotechnology. While ILs are known to stabilize or destabilize proteins, their molecular mechanisms remain poorly defined, particularly regarding effects on the unfolded ensemble. Here, we use the metastable SH3 domain of the Drosophila adaptor protein Drk, which coexists in a slow two-state equilibrium between folded and unfolded states, as a model to dissect how ILs modulate protein conformational equilibria. Using nuclear magnetic resonance (NMR) spectroscopy, we show that cholinium glutamate ([Ch][Glu]) stabilizes the folded state by preferential exclusion from the protein surface, raising the barrier to unfolding in a manner reminiscent of osmolytes and crowding agents. In contrast, 1-butyl-3-methylimidazolium dicyanamide ([Bmim][dca]) shifts the equilibrium toward the unfolded state, not by destabilizing the native fold, but by stabilizing a compact, non-native -helical conformation within the unfolded ensemble. Notably, this concentration-dependent mechanism differs from traditional denaturants such as urea or guanidinium chloride, which promote random-coil unfolded states. Kinetic measurements further reveal that [Ch][Glu] slows unfolding, whereas [Bmim][dca] slows folding, indicating that the two ILs reshape the folding landscape in opposite directions. These findings challenge the prevailing view that cosolutes act primarily on the folded state and establish unfolded-state stabilization as a critical determinant of protein stability. More broadly, this work provides a molecular framework for understanding how ILs reshape protein folding landscapes and offers design principles for tailoring ILs as stabilizers or destabilizers in biotechnology and therapeutic applications. Significance StatementProteins exist in a delicate balance between folded and unfolded states, and cosolutes such as salts are generally viewed as acting only on the folded state. Here, we show that ionic liquids (ILs) are able to reshape this balance and uniquely modulate both folded and unfolded protein states through distinct molecular mechanisms. These results challenge the conventional view of protein stabilization/destabilization and demonstrate that unfolded-state modulation is a critical determinant of protein behavior. By establishing principles for how ILs alter protein folding landscapes, this work provides a framework for designing ILs with tailored effects, with implications for biocatalysis, protein engineering, and therapeutic formulation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/700775v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@372d3borg.highwire.dtl.DTLVardef@7a7fadorg.highwire.dtl.DTLVardef@543f4eorg.highwire.dtl.DTLVardef@1084836_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Targeted protein degradation (TPD) is a powerful strategy for controlling protein abundance. Here, we establish FBXO31 as a TPD-competent E3 ligase by exploiting its recognition of C-terminal amide-bearing degrons. Using an amidated Ala-Phe motif as a chemical recruiter, multiple small-molecule binders can be transformed into FBXO31-dependent degraders that induce rapid and potent target degradation. Mechanistic studies confirm FBXO31-mediated ternary complex formation and identify key residues in FBXO31 required for recruiter engagement and target degradation. We further show that an FBXO31-based multi-kinase degrader exhibits a distinct and broader degradation profile than a CRBN-based degrader, highlighting a potentially expanded degradable target space beyond CRBN.

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.

Providing immediate access for functional proteins inside living cells would unlock unprecedented control over cellular processes; however, commonly used endocytic delivery suffers from endosomal trapping and degradation. One of the most powerful non-endosomal delivery methods uses cell surface anchored cell penetrating peptide (CPP)-additives that allow proteins to enter cells directly. Nevertheless, the underlying molecular mechanism involved in direct entry via crossing the cell membrane (protein translocation through the cell) and the major driving forces remain controversially discussed. Here, we provide a stepwise molecular picture on how CPP-additives enable uptake of protein cargoes through direct membrane translocation. CPP-additives accumulate on the cell surface in nucleation zones, locally hyperpolarizing the membrane, and induce transient water pores that allow selective CPP-protein entry without compromising membrane integrity. These fundamental mechanistic insights provide a firm basis for rationally optimizing delivery strategies using highly cationic CPPs, ultimately resulting in innovative and smart protein delivery strategies to advance therapeutic protein applications. Abstract Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/707441v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@14c1386org.highwire.dtl.DTLVardef@195f765org.highwire.dtl.DTLVardef@a52258org.highwire.dtl.DTLVardef@171dd7c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Multinuclear metal centers catalyze some of the most challenging oxidative transformations in biological and chemical catalysis, yet the molecular principles controlling oxygen activation in non-heme diiron enzymes remain unclear. Structural studies of desaturase-like diiron enzymes frequently reveal elongated Fe-Fe separations lacking canonical bridging motifs, whereas spectroscopic measurements indicate substantial metal-metal coupling during catalysis, creating a longstanding mechanistic paradox between structural and spectroscopic observations. Here we show that oxygen activation can be dynamically regulated by fluctuations in metal-metal distance through extensive multiscale simulations of the membrane-bound fatty-acid decarboxylase UndB. UndB catalyses the conversion of naturally abundant free fatty acids into terminal 1-alkenes-valuable hydrocarbon precursors for sustainable biofuels, and exhibits unusual hydrogen peroxide formation that has led to conflicting mechanistic proposals. Combining quantum mechanics/molecular mechanics (QM/MM) simulations with free energy analysis and quantum chemical calculations, we identify a catalytically competent ensemble in which transient Fe-Fe contraction establishes effective metal-metal coupling and enables formation of a peroxodiiron(III/III) intermediate despite elongated resting-state structures and the absence of canonical bridging motifs. The computed free-energy landscape quantitatively reproduces experimentally inferred catalytic barriers and accounts for the substantial H2O2 formation observed during catalysis. The reactive state resembles a P-like intermediate rather than the canonical diamond-core Q species commonly invoked for related diiron enzymes. Comparison with magnetic coupling and mechanistic features reported for related non-heme diiron enzymes, including Alkane monooxygenase AlkB, Stearoyl-CoA Desaturase 1 (SCD1) and the soluble decarboxylase UndA, suggests that dynamic metal-metal distance modulation represents a general mechanism governing oxygen activation across diverse diiron enzyme families. These findings establish metal-metal distance modulation as a previously unrecognized control parameter for oxygen activation in diiron and related multinuclear metalloenzymes, reconciling structural and spectroscopic observations and revealing how protein conformational dynamics regulate electronic coupling to control oxidative catalysis, thereby suggesting a general principle for tuning reactivity in multinuclear catalysts.

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.

Protein-based quantum sensors provide atomic-level sensitivity and precise measurements of local environments, where quantum-enabled magnetic detection can be linked to an optical readout of flavin radical pair photochemistry. Yet, the structural basis for the differing magnetosensitivities of individual proteins is still unclear, particularly regarding the respective roles of charge separation termination, complex stability, and spin relaxation. In this work, we employ all-atom molecular dynamics, quantum chemical energy calculations, Marcus-type free energy profiles, and spin relaxation theory to connect structure, electrostatics, hydration, and dynamics in AsLOV2-derived variants. Molecular dynamics simulations show that the LOV2 fold and FMN-binding core are preserved in all constructs, with enhanced flexibility restricted to surface regions, pointing to local reorganization of the donor microenvironment rather than a global loss of structural integrity. Analysis of dipolar couplings indeed demonstrates variant-specific, anisotropic inter-spin arrangements and substantially slower dephasing of the dipolar tensor, with correlation times increasing from a few nanoseconds to tens of nanoseconds. Energy gap calculations indicate strongly exergonic back electron transfer in all variants, while geometric considerations influence the differences in recombination rates. Collectively, these findings establish first principles for mechanistic design rules of engineered robust protein-based quantum sensors.

Enzymes used on industrial scale are routinely engineered for best performance. However, exhaustive mutagenesis campaigns using the twenty canonical proteinogenic amino acids rapidly reach an evolutionary ceiling, where gains in activity compromise other critical properties such as thermal endurance. Although non-canonical amino acids (ncAA) expand the chemical space, most are costly for use on an industrial scale and significantly perturb structure. Here, we demonstrate that the evolutionary ceiling of highly optimized polyethylene terephthalate (PET) hydrolases (PETases) can be broken with azatryptophans that (i) differ minimally from their canonical tryptophan, (ii) are genetically encoded, and (iii) are produced in high yield by enzymatic biosynthesis from inexpensive precursors. The first genetic encoding systems are described for 4-azatryptophan, 5-azatryptophan, and 6-azatryptophan, achieving single, site-selective isosteric CH [->] N substitutions that enhancing the catalytic activity while preserving thermal stability. The fluorescence of 6AW provides a uniquely sensitive reporter of side-chain solvent exposure, which is critical for PETase activity and shown to vary between five different PETases. Furthermore, Azatryptophan-bearing enzymes are inexpensive to produce. To benchmark PETase activity, a rapid fluorescence-based kinetic assay, PETra, is introduced, which delivers consistency and reproducibility by using a soluble substrate yet correlates strongly with the hydrolysis of solid PET.