Metal-dependent and metal-free mechanisms of peptide condensate catalysts

Domain: physics
Published: 2026-03-28
Editorial outcome: major_revision

Abstract
Condensates formed via liquid-liquid phase separation (LLPS) provide a chemically versatile environment for catalysis through dynamic molecular interactions. We present designed biomolecular condensates, formed by LLPS of minimalistic histidine-containing peptides, catalyzing ester hydrolysis with two distinct mechanisms. Zn2+-dependent condensates activate a coordinating water molecule at the active site, formed by Zn2+-histidine coordination, enabling nucleophilic attack. We show that dense-phase basicity, internal mobility, and Zn2+ accumulation within the condensates collectively govern their catalytic activity. In the absence of Zn2+, catalysis is driven by intermolecular low-barrier hydrogen bonds between histidine residues, facilitating nucleophile formation. Combined computational and experimental evidence reveals the molecular basis of these catalytic pathways, demonstrating the functionality of biomolecular condensates in catalysis and nanotechnology. These findings establish a foundation for exploring mechanisms of metal-free emergent catalysis within complex liquid assemblies, expanding the potential of LLPS-based systems in green chemistry and advanced materials.

Introduction
Enzymes facilitate a diverse range of reactions under mild aqueous conditions with exceptional efficiency and specificity. Drawing inspiration from enzymes, researchers have developed supramolecular catalysts1, utilizing self-assembled building blocks2,3 to construct complex structures with emergent and tunable properties4–6. Peptides have gained prominence as supramolecular catalytic building blocks due to their biocompatibility, natural presence in enzymatic catalytic sites, and chemical versatility2,4,5,7. Peptide-based self-assembled functional materials, including α-helix,8 hydrogels9/fibrils10,11, nanotubes12 and nanowire superstructure13 have been developed as artificial biocatalysts2–5,7,10,12,14–17. These assemblies mimic diverse enzymatic capabilities, including peroxidase18, aldolase19/retro aldolase20, and hydrolase11,16 activities. Hydrolases, particularly esterase- and phosphatase-mimetic supramolecular systems, have been extensively studied for their applications in drug delivery, agriculture, and environmental remediation15.

The active sites of hydrolases frequently contain histidine (His) residues, which play a central role in catalysis. His may function as ligands for metal ion cofactors, participate in catalytic dyads or triads, or mediate reactions through interactions with other His residues21. In esterase mimics, His residues often coordinate Zn²⁺ ions, providing structural stability and catalyzing ester hydrolysis13,16,22. Computational studies have shown that three His residues can coordinate Zn²⁺ ions, with a fourth His residue accepting the excess proton23,24. Alternatively, His residues can catalyze ester hydrolysis without Zn²⁺ ions10,11,25, wherein adjacent His residues are both protonated and deprotonated participate in the catalysis26.

Moving from hierarchical supramolecular structures to disordered liquid assemblies, the emergent field of liquid-liquid phase separation27 (LLPS) provides opportunities to design dynamic and tunable environments. Inspired by membraneless organelles28, biomolecular condensates are dense phases formed via LLPS of biomolecules27, driven by multivalent, weak non-covalent interactions. A decade of research into the amino acid or nucleic acid interaction modules underlying LLPS27,29 has enabled the design of condensates30 based on various building blocks, including engineered proteins and polypeptides31, DNA32/RNA33 and short peptides34–37. The chemical diversity and sequence programmability of designed condensates allow precise control over their formation and properties.

In addition to their ability to sequester a broad range of molecules, including large macromolecules and small hydrophobic compounds, condensates have been employed in various applications mimicking cellular organelles, such as microreactors37–41, cell-free transcription-translation systems42, and intracellular delivery43. More recently, the spatiotemporal regulation of chemical reactions by condensates suggest their potential to function not only as vessels for chemical reactions37,39,44 but also as active catalytic entities45–49.

Designing condensates as catalysts can be achieved through various strategies, including the fusion of enzymes to low complexity domains (LCDs) that promote phase separation49, utilizing intrinsically disordered proteins47,48, employing short peptides alone46 or with other building blocks45. These condensates enhance catalysis through interfacial electric potentials47,48, distinct intrinsic environment45,46,49 and partitioning46. Despite these advancements, many questions remained unanswered about the molecular mechanisms underlying the catalytic capabilities of condensates.

Here, we introduce a class of catalytic condensates formed through complex coacervation of His-containing peptides. The formation of condensates, along with their catalytic ability to enhance ester hydrolysis, are mediated by two independent pathways: a metal-dependent mechanism and a metal-free mechanism. In the metal-dependent pathway, Zn2+ ions coordinate with the peptides and active their bound water molecules to generate nucleophilic hydroxide groups, while in the metal-free pathway, intermolecular low-barrier hydrogen bonds (LBHB) between His residues enhance nucleophile formation.

Our findings shed light on the molecular mechanisms underlying catalytic condensates and connect the condensates materials properties with their catalytic capacity emphasize the potential of phase-separating liquid materials in regulating reactivity. Furthermore, they highlight promising opportunities for designing supramolecular liquid-based biocatalysts through precise molecular engineering, a process expected to be further accelerated with the integration of machine learning50 tools in the near future.

Results
Peptide LLPS is mediated through coordination to Zn2+ ions
We sought to develop a minimalistic condensate system with catalytic capacity by utilizing LLPS-promoting peptides through the incorporation of His as catalytic moieties. We hypothesized that these condensates could enhance ester hydrolysis due to their dynamic character and His-rich catalytic site. We proposed that the dense phase of the condensates might facilitate substrate partitioning while molecularly enriched environment that enables multiple modes of peptide-peptide intermolecular interactions within the dense phase, where the catalytic moieties promote the substrate hydrolysis (Fig. 1a). The condensates are formed by two oppositely charged peptides, which builds on our recent design of minimalistic LLPS-promoting peptides that assemble into biomolecular condensates36. We incorporated either tyrosine (Tyr) or one or two His, separated by Gly, at the C-terminus of the peptide to generate a small library of His-containing LLPS-promoting peptides (Fig. 1b).

We analyzed the LLPS propensity of oppositely charged peptides with varying number of His: R2H/E2H, R1H/E1H and R/E (Fig. 1). Building on previous work on esterase-mimicking catalytic peptides16, we used Zn2+ as a cofactor for ester hydrolysis and hypothesized that Zn2+ could function both as a cofactor and an initiator of LLPS (Fig. 1). We created phase diagram heat maps by monitoring sample turbidity as a factor of peptide concentration (Fig. 2a-c, Supplementary Tables 2-4) and confirmed condensate formation by bright-field microscopy (Supplementary Fig. 1).

Phase diagrams showed an inverse relationship between the number of His residues and the saturation concentration (C , Supplementary Fig. 1), suggesting that His enhances LLPS through specific interactions with Zn2+ ions. Replacement of ZnCl with CaCl or NaCl confirmed that LLPS is driven by Zn²⁺ coordination, alongside peptide-peptide interactions (Supplementary Fig. 2). Previous studies also support the role of divalent metal ions, particularly Zn2+, in mediating phase separation51,52. Moreover, the effect of ZnCl on LLPS is concentration-dependent (Supplementary Fig. 2), where 0.2 mM is the critical concentration to trigger LLPS. Thus, ZnCl concentration directly modulates the dense-phase concentration of the R2H/E2H peptides. Increasing the ZnCl concentration from 0.2 and 0.33 mM to 0.67 mM resulted in 1.7-fold and 2.2-fold increases, respectively, in the total peptide concentration within the dense phase (Fig. 2d). Dense-phase peptide concentrations were determined by quantifying the peptide concentration remaining in the dilute phase following droplet centrifugation, using absorbance spectroscopy calibrated for the peptides. These findings indicate that Zn2+ concentration may also play a critical role in influencing the catalytic capacity of the condensates.

The catalytic performance of the condensates is restricted by Zn2+ ions
We aimed to analyze the catalytic capacity of the peptide condensates, focusing on the His-rich R2H/E2H system at 0.5 mM. To this end, we selected 4-methylumbelliferyl acetate (4-MU-Ac) as the model ester substrate for hydrolysis (Supplementary Fig. 3), which produces the fluorescent product 4-methylumbelliferone (4-MU). Due to the low solubility of 4-MU-Ac and 4-MU in aqueous solutions, the selected concentration range was limited to 1.1 mM, resulting in linear initial velocity (V ) plots (Fig. 2e). To consider the condensates' impact on 4-MU's fluorescence intensity, we used calibration curves in condensate solution (Supplementary Fig. 4) and confirmed that 2.8% MeCN, used to dissolve 4-MU-Ac, did not affect condensate formation (Supplementary Fig. 5).

We evaluated the catalytic performance and product formation (Supplementary Fig. 6) in the presence of ZnCl (0.67 mM). The V value (2.0E-05±7.0E-5) was found >6.5-fold higher than that in bulk buffer in the presence of Zn2+ at substrate concentration of 0.7 mM (Supplementary Table 5). Each of the peptides independently, R2H and E2H, does not undergo LLPS yet affect the reaction kinetics, where the V is ~2-fold and 3.7-fold faster under LLPS conditions (R2H/E2H) compared to when no LLPS is observed (R2H or E2H alone) (Fig. 2e, Supplementary Table 5). Notably, dynamic light scattering (DLS) analysis showed that the soluble R2H and E2H peptides form nanometer-sized assemblies (Supplementary Fig. 7). The condensates formed following complexation of R2H and E2H have an average size of 1.2 ± 0.25 m. This observed V value of R2H/E2H with 0.67 mM Zn2+ is lower than those previously reported for Zn2+-dependent self-assembling catalysts with ordered structures for p-nitrophenyl acetate (pNPA) hydrolysis3,16,22. Yet, decreasing the Zn²⁺ concentration to 0.33 mM and 0.20 mM resulted in 2.6-fold and 3.4-fold increase in the V at 0.7 mM 4-MU-Ac compared to 0.67 mM Zn²⁺, respectively (Fig. 2e). This unexpected trend was also observed in a previous study53, however, most studies on metal-dependent self-assembled peptide catalysts report on acceleration of reaction kinetics mediated by Zn2+ ions16,22.

To further elucidate how Zn²⁺ concentration influences the catalytic activity of the phase-separated systems, we measured the pH of both the dilute and dense phases using the ratiometric pH probe SNARF-1 and by confocal laser scanning microscopy (CLSM)48,54,55. The emission ratio of SNARF-1 at λ = 580 and 640 nm varies as a function of pH (Supplementary Fig. 8). Calibration curves were obtained by measuring the I ratio in buffers of defined pH containing varying Zn²⁺ concentrations, enabling quantitative determination of pH in each phase (Fig. 2f–g). The pH of the dilute phase was similar across all systems, with 7.7 ± 0.11 and 7.6 ± 0.40 for 0.2 mM and 0.67 mM Zn²⁺, respectively. In contrast, the dense-phase pH was strongly dependent on Zn²⁺ concentration, decreasing from a highly basic value of 10.8 ± 0.60 at 0.2 mM Zn²⁺ to 8.5 ± 0.10 at 0.67 mM Zn²⁺ (Fig. 2f–g, Supplementary Table 6). We further analyzed how Zn2+ concentration affect the internal mobility of the dense phase using fluorescence recovery after photobleaching (FRAP) by CLSM. For this, we used rhodamine b as a fluorescent payload and probe. By tracking the recovery of rhodamine b fluorescence following photobleaching (Fig. 2h-j) we obtained the t values for condensates with increasing Zn2+ concentrations (Fig. 2j). While all three systems exhibit condensate coalescence characteristic of LLPS (Supplementary Videos 1-3), increasing Zn²⁺ concentration leads to distinct differences in diffusivity. We note that the absolute diffusivity values obtained using rhodamine B may be influenced by electrostatic dye–peptide interactions. At 0.67 mM Zn2+, only 35.5 ± 11.7% recovery of rhodamine b signal was observed, indicating arrested solid-like dense phase with restricted diffusion (Fig. 2h-i). Lowering the Zn2+ concentration to 0.33 mM and 0.2 mM, resulted in increase in the recovery after photobleaching to 81.1 ± 6.3 % and 84.9 ± 5.2%, respectively (Fig. 2i), with t values of 48.8 ± 7.7 s and 36.0 ± 5.0 s for 0.33 mM and 0.2 mM Zn2+ (Fig. 2j). Additionally, we analyzed the Zn content within the dense phase of droplets formed at 0.2, 0.33, and 0.67 mM ZnCl₂ using scanning transmission electron microscopy (STEM) coupled with energy-dispersive X-ray spectroscopy (EDS). High-angle annular dark-field (HAADF) imaging combined with EDS line profiling enabled comparison of the relative Zn signal within the dense phase (Fig. 2k). This analysis revealed a pronounced enrichment of Zn at higher Zn²⁺ total concentrations, with approximately 1.5-fold and 5-fold increases in Zn intensity in droplets formed at 0.67 mM Zn²⁺ compared to those formed at 0.33 mM and 0.2 mM Zn²⁺, respectively (Fig. 2k). The partitioning of the 4-MU product was weakest in condensats formed at 0.67 mM Zn²⁺ (Supplementary Fig. 9), likely due to the highly crosslinked dense-phase network at this Zn²⁺ concentration, which reduces accessibility and limits internal porosity.

The difference in dense-phase pH at varying Zn2+ concentration can be attributed to Zn²⁺-His coordination. At lower Zn²⁺ concentrations, fewer His residues are coordinated, increasing the effective basicity of the dense phase. At higher Zn²⁺ concentrations, extensive Zn²⁺-His coordination reduces the availability and basicity of free His residues, resulting in a lower dense-phase pH. The elevated basicity observed at low Zn²⁺ is expected to promote the hydrolytic reaction, consistent with enhanced reaction rate measured in bulk buffer under basic conditions (Supplementary Fig. 10) and provides a mechanistic basis for the highest catalytic activity observed at 0.2 mM Zn²⁺. FRAP measurements further demonstrate that increasing Zn2+ markedly attenuate molecular diffusivity within the dense phase, likely due to Zn2+-mediated physical crosslinking of the peptide network. This limited diffusion and mass transfer at higher Zn2+ concentrations can contribute to the inhibitory effect of Zn2+ on catalytic performance.

Collectively, these results indicate that dense-phase basicity, internal mobility, and Zn accumulation within the condensates act in concert to regulate catalytic activity (Fig. 1c).

Molecular level studies of Zn2+-mediated catalytic capacity of condensates
To further understand the mechanistic role of Zn2+, we constructed an atomic model of the condensate R2H/E2H and carried out classical molecular dynamics (MD) simulations. The pair distribution function of His C atoms calculated from trajectories showed a significant peak in the range of 6.5-7.5 Å (Supplementary Fig. 11). Based on this, we designed a catalytic center consisting of two peptides, a Zn2+ ion, neighboring water molecules, and the substrate, where two His residues from one peptide and one His residue from the other peptide coordinates the Zn2+. In this model, the distance between the C atoms of the two His residues is 6.67 Å, consistent with those obtained from the MD simulations. Then, we performed ab initio calculations to obtain a stepwise reaction mechanism.

As detailed in Fig. 3, the reaction proceeds in four steps: first, Zn2+ activates its bound water molecule, and a proton transfers from the water to the C-terminus to form a hydroxide group in complex 1, with a free energy 3.2 kcal/mol higher than complex 1’. Then, the substrate approaches, and the hydroxide ion nucleophilic attacks the carbon atom of the substrate’s ester bond, with a free energy barrier of 20.8 kcal/mol. This interaction initiates the formation of a new C-O (hydroxyl) bond (from 2.73 Å in compound 2 to 1.46 Å in compound 3) through transition state TS2/3. Concurrently, the substrate’s ester bond C-O distance extends from 1.38 Å to 1.52 Å in compound 3, indicating a propensity for cleavage.

Intermediate 3 is followed by breaking the ester bond with a free energy barrier of 16.0 kcal/mol (TS3/4), forming acetic acid and 4-methylumbelliferone phenolate ion in complex 4. The latter can easily take a proton from the uncoordinated His, mediated by two water molecules, ultimately forming the fluorescent alcohol 4-MU. The free energy of the resulting complex 5 is 4.4 kcal/mol lower than that of complex 2. In the last step, the product dissociates from the catalytic center, and a new water molecule enters the catalytic center (complex 6), with a free energy change of -2.0 kcal/mol. Following this, a proton from the Zn2+ bound water molecule transfers to the deprotonated His via mediation by water molecules, regenerating the catalytic center structure as in complex 1 and initiating the next cycle.

The free energy barrier of 20.8 kcal/mol for the rate-determining step is fairly comparable with free energy barriers reported for pNPA hydrolysis by catalytic self-assembled structures13,23, and lower than others3, suggesting that the catalytic reaction can occur spontaneously at room temperature.

To confirm that the peptide directly interacts with Zn2+ ions, we performed CD analysis in the absence or presence of ZnCl . The CD analysis (Supplementary Fig. 12) shows that the His-containing peptides R2H and E2H retain a random-coil signature with a ~196 nm minimum and a ~225 nm maximum yet show distinct spectral shifts upon addition of ZnCl₂. For E2H, a new maximum near 203 nm appears and intensifies with increasing Zn²⁺ concentration, while R2H displays a progressive decrease in its random-coil minimum under the same conditions. In contrast, peptides lacking His show no detectable spectral changes in the presence of Zn²⁺, indicating that the metal interacts specifically with His-containing peptides. In addition, we leveraged the intrinsic fluorescence of Trp within the peptides to perform Trp emission analysis in which environmental changes to the indole side chain results in changes in the emission intensity ( =280 nm). The results show a significant increase in the Trp emission intensity for the His-containing peptides, R2H and E2H, in the presence of 0.67 mM ZnCl while no change is observed for the peptides lacking His, R and E (Supplementary Fig. 12).

Together, the CD and Trp emission analysis show that Zn2+ ions interact with the His-containing peptides. These observations strengthen the phase diagrams and FRAP results which show that Zn2+ promotes phase separation and slows the dense phase internal mobility, likely by strong interactions with R2H and E2H.

Zn2+-free formation of biomolecular condensates
In light of the inhibitory effect of Zn2+ on condensate catalytic capacity, we next examined catalysis in the absence of ZnCl as an alternative mechanistic regime. We first analyzed the LLPS propensity of oppositely charged peptides (at 1:1 ratio) in the absence of ZnCl . Structural characterization revealed that without Zn2+, no condensates are formed by R2H/E2H and R1H/E1H within the same concentration range used in the Zn2+-mediated system (Fig. 4a), even after increasing the peptide concentration tenfold. Subsequently, we analyzed the LLPS propensity of various combinations of the positively charged His-containing peptide (R2H, R1H) with the negatively charged peptide (E). LLPS was observed for all combinations with E, at a concentration of 3 mM (Fig. 4a). In contrast to the trend observed in the Zn2+-dependent system, the R/E system exhibits the highest LLPS propensity (Fig. 4a). Similarly, the dense-phase peptide concentration of R/E is 1.5-fold higher than that of R2H/E (Fig. 4b) further showing the higher efficiency of phase separation in this system. This suggests that the Zn2+-mediated and the Zn2+-free LLPS are driven by different interactions, with the former likely facilitated by interactions between His or Glu residues and the Zn2+ ions56. These observations indicate that LLPS is mediated by a complex interplay of intermolecular attractive forces rather than solely electrostatic interactions, where Tyr plays a distinctive role in promoting LLPS36.

MD simulations of Zn2+-free LLPS systems show that the R2H/E system gradually aggregates from the initial random distribution to form a condensed, water-containing phase (Fig. 4c). We measured the aggregation propensity of different peptides by calculating the degree of collapse, defined as the ratio of the solvent-accessible surface area (SASA) during the simulation to that of the fully random state in the initial state, where higher collapse degree indicates tighter peptide packing. As shown in Supplementary Fig. 13, in molecular dynamics simulations, the formation of peptide condensates reached equilibrium after approximately 200 ns. The R2H soluble peptide, which does not phase separate by itself, shows a lower collapse degree than R2H/E (Fig. 4d), indicating its higher water solubility. We defined the solvent-accessible surface of the peptide condensate as the boundary and calculated the enclosed volume and the number of water molecules within this boundary to determine the water content in the condensate during the simulation. The water density of the different systems is consistent with their collapse degree (Fig. 4e). Time-lapsed microscopy analysis showed that the R2H/E and R/E condensates coalesce and fuse over time, confirming their nature as hydrated liquid assemblies (Supplementary Videos 4-5).

Zn2+-free catalytic performance of peptide condensates
We determined product formation (Supplementary Fig. 14) and V for each system as described above by using specific calibration curves of 4-MU (Supplementary Fig. 15), focusing on the lowest possible concentration of the peptides which undergo LLPS (3 mM) without ZnCl . In the absence of Zn2+ ions, the His-containing condensates demonstrate a remarkable ability to enhance catalysis as a function of His content (Fig. 5a). At 0.7 mM 4-MU-Ac the V of R2H/E is 4.2-fold higher than that of R2H/E2H with 0.20 mM ZnCl demonstrating the Zn2+-free condensates superiority over the Zn2+-dependent condensates (Fig. 1). Therefore, we calculated the kinetic parameters for these systems (Supplementary Table 7). Among the three systems tested (R2H/E, R1H/E and R/E), the R/E system shows minimal increase in V values with increasing substrate concentration (Fig. 5a) and exhibits the lowest V and k values (Supplementary Table 8). In contrast, the k value of R2H/E and R1H/E is 3.5 and 7.0-fold higher than that of R/E. At 0.7 mM 4-MU-Ac, the V of the R2H/E condensates is 1.3-fold and 6.9-fold higher than that of R1H/E and R/E, respectively (Fig. 5a, Supplementary Table 8). After 2.5 min R/E showed <2% conversion, highlighting the critical role of His in catalysis. Moreover, when monitored for 60 min, R2H/E and R1H/E reach a plateau of fluorescent signal, while R/E is still in the linear phase (Supplementary Fig. 16).

To dissect the determinants of condensate activity, we tested whether replacing Arg with Lys would alter LLPS propensity. Strikingly, K2H/E did not undergo LLPS even at high peptide concentrations (10 mM; Supplementary Fig. 17), highlighting the critical role of Arg-mediated interactions in driving phase separation36, whereas His-Tyr interactions alone are insufficient. CLSM imaging showed progressive accumulation of 4-MU fluorescence within R2H/E condensates over the course of the reaction (Fig. 5b). To analyze partitioning, 4-MU was first dissolved in MeCN and then added to preformed R2H/E droplets such that the final MeCN concentration was 2.8%. Under these conditions the fluorescence intensity of 4-MU in the dense phase was 24.3-fold higher than in the dilute phase (Fig. 5c).

To elucidate the catalytic contribution of LLPS, we conducted control kinetic experiments with 0.7 mM 4-MU-Ac. The V of the R2H/E and R1H/E condensates were 81- and 63-fold higher, respectively, than spontaneous ester hydrolysis in buffer (Supplementary Table 8). Supernatants collected from centrifuged LLPS samples displayed reduced activity (1.8- and 1.7-fold lower than the two-phase systems), (Supplementary Table 8), and contained small heterogeneous microscopic structures, suggesting that residual assemblies may contribute to catalysis. Next, we compared condensates with each of the peptides alone, as a control. At the same peptide concentration (3 mM), individual R2H or E peptides—unable to undergo LLPS—displayed only slightly reduced V₀ compared to the condensates (Supplementary Table 8). Moreover, R2H and R1H peptides showed markedly higher V values compared to the peptide R which lacks His (16.9- and 14.8-fold above R, respectively), confirming the essential role of His in promoting hydrolysis. Yet, this effect was not attributable to free His alone, since 3 mM His displayed far lower catalytic activity–5.0- and 4.4-fold lower than R2H and R1H, respectively (Supplementary Table 8). To probe the structural basis of this activity, we used DLS and transmission electron microscopy (TEM) to test whether the R2H and R1H alone form nanoscale assemblies. Indeed, R2H and R1H produced nanostructures averaging 281.3 ± 92.4 nm and 395.1 ± 154. 4 nm in diameter, respectively (Fig. 5d). Addition of E triggered LLPS, resulting in micron-sized droplets with an average diameter of 1.1 ± 0.43 m for R2H/E, 1.3 ± 0.42 m for R1H/E, and 2.2 ± 0.30 m for R/E (Fig. 5d). TEM confirmed the formation of amorphous nanometric assemblies by R2H (Supplementary Fig. 18), consistent with previous reports that such supramolecular structures enhance hydrolysis110,11. Despite their catalytic activity, the soluble peptides displayed a propensity to form low-abundance crystals. Bright-field imaging of R2H revealed elongated crystals emerging within minutes of substrate addition (0.7 mM, t=3 min; Supplementary Fig. 19a). In contrast, condensates suppressed crystal formation, even after prolonged reaction times (15 min; Supplementary Fig. 19b), suggesting that sequestration of substrate and/or product within the dense phase prevents crystallization.

To shed light on the catalytic capacity of the condensates, we measured the pH of the dense and dilute phases using the ratiometric probe SNARF-1. Both phases in the R2H/E and R/E systems were found to be basic, with dilute-phase pH values of 8.0 ± 0.1 and 8.4 ± 0.3, respectively (Fig. 5e-f, Supplementary Table 6). The basic environment in both phases is expected to promote the hydrolytic reaction, consistent with the enhanced catalytic activity observed in bulk buffer under basic conditions (Supplementary Fig. 16). FRAP analysis of R2H/E and R/E condensates (Fig. 5g) suggested that both condensates are liquid assemblies (Fig. 5h) that exhibit typical coalescence (Supplementary Videos 4-5), with almost full recovery after photobleaching (95.7 ± 2.6% and 99.9 ± 2.6% for R/E and R2H/E, respectively). Yet, R2H/E condensates had 4.7-fold faster t value than that of R/E condensates (1.4 ± 0.3 sec for R2H/E and t =6.5 ± 1.5 sec for R/E) (Fig. 5i). Together, these results suggest that the enhancement in ester hydrolysis is amplified by phase separation through substrate partitioning, elevated local concentrations, and suppression of inhibitory crystallization. Tyr-driven LLPS is essential for forming dynamic, liquid-like condensates, while His content governs catalytic efficiency. Thus, optimal catalysis emerges from the interplay between peptide sequence, supramolecular assembly, and condensate microenvironment.

The catalytic performance of Zn2+-free condensates is mediated by LBHB
MD simulations indicated that intermolecular hydrogen bonding between His residues is probable within the condensed phase (Fig. 5j-k). A detailed analysis of the C atom distance distributions between His residues from distinct peptides shows that, within the interaction range where His side chains can interact (C atom distances of ~8 Å), R2H/E exhibits a higher frequency than R1H/E (Fig. 5j-k). LBHB formation by different N /N , configurations on the imidazole ring (Fig. S22). In the first step, a neighboring water molecule donates a proton to one His via a water bridge, leading to formation of a hydroxide group and a positively charged His (complex 2’), with a free energy change of +8.0 kcal/mol. Consequently, the two His then form a LBHB in the “imidazolium-imidazole” configuration, as characterized by the markedly short distance (~2.58 Å) between the donor and acceptor nitrogen atoms, and the negligible free energy barrier of only 0.7 kcal/mol (TS2’/1). The transfer of the shared proton further stabilizes the hydroxide ion.

Once stabilized, the hydroxide ion attacks the ester carbon of the substrate, initiating bond cleavage with a free energy barrier of 14.2 kcal/mol (TS2/3). Upon the cleavage of the ester bond, acetic acid and phenol are produced (complex 4 ; free energy change of 13.9 kcal/mol), followed by proton transfer to generate the 4-MU. In the final step, rearrangement of the hydrogen bond network between water molecules (TS6/7) resets the system, preparing the system for another catalytic cycle. The rate-determining step is the nucleophilic attack on the ester bond, with a free energy barrier of 14.2 kcal/mol, suggesting that this process can occur at room temperature. Notably, this barrier is lower than in the Zn2+-dependent system, providing a mechanistic explanation for the enhanced catalytic efficiency of the Zn2+-free condensates (Figs. 2e, 5a).

Lastly, we modulated the protonation states of His residues within the R2H/E system to varying degrees in MD simulations (Supplementary Table 9). Considering that the pKa for the complete deprotonation of His to form its anionic form is approximately 13, we only consider its neutral and cationic forms. We compared the collapse degree and the pairwise His’ Cα-Cα distance distribution across neutral and partially protonated systems (Supplementary Fig. 23). The results demonstrate that His state does not significantly alter the LLPS properties of the peptide droplet (degree of collapse). Furthermore, the Cα distance distribution remained largely unaffected, suggesting that the structural prerequisites for LBHB formation are maintained. These results suggest that while the local acid-base environment within the droplet may differ from that of the bulk phase, our proposed molecular mechanism remains relatively robust to pH variations. This stability might be attributed to histidine partially acting as a buffer agent, effectively maintaining the internal structural integrity of the LLPS under varying solution conditions.

Methods
Materials
Peptides were custom synthesized, then purified by high performance liquid chromatography to 95% and supplied as lyophilized powders by Genscript, Hong Kong. Unless otherwise specified, all reagents were of the highest available purity. 4-Methylumbelliferyl acetate was purchased from Tzamal D-Chem. 4-Methylumbelliferone was purchased from Rhenium. 5(and 6)-Carboxy SNARF 1 was purchased from LabSuit Shop. Acetonitrile, NaCl, NaOH and HCl were purchased from BioLab. ZnCl , Sigmacote and Trizma base were purchased from Merck.

Phase diagrams and turbidity measurements
Zn+2-mediated phase diagram:
Stock solutions of positively charged peptide (solution A) and negatively charged peptide (solution B) were prepared at 1.5 mM in a 0.67 mM ZnCl solution in 10 mM Tris-HCl and the pH of each solution was adjusted to 7.50 ± 0.04. Samples were formed by adding buffer, solution A and solution B, at final concentrations of 0.05, 0.01, 0.25, 0.5 mM. For R/E system the following concentrations combinations were not evaluated due to the results obtained for R1H/E1H at the same conditions: 0.01/0.01, 0.01/0.05, 0.01/0.25, 0.01/0.5, 0.05/0.01, 0.25/0.01, 0.5/0.01.

Turbidity measurements
The turbidity of 36 μl of each condition was measured in quadruplicates in room temperature at λ=350 nm using a Synergy H1 or Epoch2 microplate reader. For each condition, buffer background was subtracted from the average absorbance. Value s below 0 were defined as 0. All experiments were performed in two independent measurements. Data represents the corresponding intensity for the displayed images.

Zn+2-free phase diagram
Stock solutions of solution A and solution B were prepared at 12 mM in10 mM Tris-HCl and the pH of each solution was adjusted to 7.50 ± 0.04. Samples were formed by adding buffer, solution A, solution B and MeCN (final concentration of 2.8%), at final concentrations of 0.75, 1.5, 3, 5 mM.

Microscopy analysis of LLPS
For both systems, the samples were imaged (50 µl) immediately after preparation in a 96-well black glass bottom plate, by fluorescence microscope (Olympus), x40/0.95 NA Plan Extended Apochromat objective. Images were collected and processed using CellSens Dimension software. Display ranges were adjusted individually for clarity of visualization.

Effect of ZnCl and NaCl on LLPS
All ZnCl -containing buffer solutions were prepared from an initial stock of 10, 7.68 or 1.67 mM ZnCl in 10 mM Tris-HCl pH 7.50 ± 0.04, at final concentrations of 0.1, 0.2, 0.3 and 0.4 mM ZnCl . For CaCl and NaCl analyses, peptides stock solutions were prepared in 0.67 mM salt in 10 mM Tris-HCl buffer. Stock solutions of R2H and E2H were prepared at 1.5 mM (final concentration of 0.5 mM of each peptide) in an appropriate buffer and the pH of each solution was adjusted to 7.50 ± 0.04. Samples were formed by adding buffer, R2H and E2H. The samples were imaged as described above.

Total peptide content in the dense phase
Calibration curves:
Stock peptides solution (positive and negative) at concentration of 1mM were prepared in 10 mM Tris buffer. The peptide solutions were mixed and dilute with buffer and 2.8% ACN to achieve final peptide concentrations of 0.1, 0.08, 0.06, 0.04 and 0.02 mM from each peptide. 36 μl of each peptide solution was added to a 384 well plate. Absorbance spectra of quadruplicates measured at range of 230-300 nm in a 384 well black plate by Biotek H1 synergy plate reader (purchased from Lumitron, Israel). The calibration curves were calculated at 322 nm.

Zn2+-dependent:
solutions of 1.5 mM E2H and R2H peptides were prepared in 10mM Tris with 0.2, 0.33 and 0.67 mM ZnCl at pH 7.5±0.04. A solution of a final concentration of 0.5 mM of each peptide with 1 mM 2.8% ACN were made at volume of 144 μl in a 1.5ml Eppendorf tube. After 10 min of incubation, the samples were centrifuged at 1,500 × g for 7 minutes. A volume of 120 μl from the supernatant was collected and diluted 4 times with 10 mM Tris buffer. The absorbance of triplicates of 36 μl of the diluted supernatant was measured at λ = 280 nm in a 384 well black plate by Biotek H1 synergy plate reader (purchased from Lumitron, Israel). All experiments were performed in triplicate. The concentration of the supernatant solutions determinate by the calibration curves.

Zn2+-free:
solutions of 9 mM positive and negative peptides were prepared in 10 mM Tris at pH 7.5±0.04. A solution of a final concentration of 3 mM of each peptide with 1 mM 2.8% ACN were made at volume of 144 μl in a 1.5 ml Eppendorf tube. After 10 min of incubation, the samples were centrifuged at 17,700 × g for 1 hour. A volume of 120 μl from the supernatant was collected and diluted 4 times with 10 mM Tris buffer. The absorbance of triplicates of 36 μl of the diluted supernatant was measured at λ = 280 nm in a 384 well black plate by Biotek H1 synergy plate reader (purchased from Lumitron, Israel). All experiments were performed in triplicate. The concentration of the supernatant solutions determinate by the calibration curves.

%EE was calculated using Eq. 1.
(1) %𝐸𝐸 = (𝐶𝑇−𝐶𝑠𝑢𝑝)/𝐶𝑇

Reaction kinetics analysis
Choice of substrate and working concentration range: We selected 4-methylumbelliferyl acetate (4-MU-Ac) because the fluorescent 4-MU product allows simultaneous kinetic monitoring and spatial imaging by confocal laser scanning microscopy—measurements that are central to the mechanistic claims in this work. Absorbance-based substrates (e.g., pNPA/pNPP) are more soluble and permit broader bulk concentration ranges, but absorbance readouts in intact two-phase samples are strongly affected by light scattering from droplets, which evolves during LLPS and cannot be reliably blanked; therefore, absorbance assays were not used on intact condensates here. We empirically selected a working substrate window of 0.3–1.1 mM as concentrations above 1.1 mM promoted rapid crystal formation in several peptide sequences and reduced reproducibility.

Stock solution (39.6 mM) of 4-MU-Ac was prepared in MeCN, lower concentrations were diluted from this stock in MeCN (32.4, 25.2, 18 and 10.8 mM, for final concentrations of 1.1, 0.9, 0.7, 0.5 and 0.3 mM respectively). Stock solutions of solution A and solution B were prepared at 1.5 mM in 0.67/0.33/0.20 mM ZnCl solution in 10 mM Tris-HCl and the pH of each solution was adjusted to 7.50 ± 0.04. Samples were prepared directly in wells. Substrate was added lastly from the appropriate stock by pipetting into the well, at a final concentration of 0.5 mM of each peptide. The fluorescence intensity was immediately measured (λ = 360 nm, λ = 450 nm) for 3 min in a 384-well black plate with a clear bottom or a UV plate, by a Biotek H1 synergy plate reader. The V was obtained by slope of data acquired up to 2.5 min using a linear fit and divided by the calibration curve slope. Soluble peptides were at 0.5 mM final concentration. The same protocol was used for Zn+2-free condensates (R2H/E, R1H/E, R/E), with appropriate peptide concentrations (final concentration of 3 mM of each peptide). Zn+2-free soluble peptides were at 3 mM final concentration. Kinetic parameters were calculated by fitting the data to the Lineweaver Burk plot using linear regression, the calculated errors were calculated using the errors obtained from the linear fit. Catalyst concentration was set as 3 mM for all systems. For the 1 h kinetics analysis, the measurements were performed for 60 min. For kinetics at pH 9 the pH of the buffer was adjusted to 9 ± 0.04. Control samples (addition of MeCN instead of substrate) were subtracted from the average fluorescence intensity value. Displayed results are one representative experiment from three independent measurements.

Conversion calculations:
For each system, control samples (addition of MeCN instead of product) were subtracted from the average fluorescence intensity value at 2.5 min, and then divided by the corresponding calibration curve’s slope. The conversion was calculated by Eq. 2:
(2) % Conversion = (product concentration [mM] / 0.7[mM]) * 100

Calibration curves
Calibration curves were prepared to each system separately.
Stock solution (7.2 mM) of 4-MU was prepared in MeCN, lower concentrations were diluted from this stock in MeCN (5.4, 2.7, 0.9, 0.225, 0.16, 0.08 and 0.04 mM for final concentrations of 0.2, 0.15, 0.075, 0.025, 0.006, 0.004, 0.002 and 0.001 mM, respectively). Peptide solutions were prepared in 10 mM Tris-HCl and the pH was adjusted to 7.50 ± 0.04. Condensate solutions were prepared directly in wells. 4-MU was added lastly from the appropriate stock by pipetting into the well. The fluorescence intensity was measured (λ = 360 nm, λ = 450 nm) in a 384-well black plate with a clear bottom or UV plate by a Biotek H1 synergy plate reader. Control samples (addition of MeCN instead of product) were subtracted from the data. Calibration curves values obtained by averaging the data acquired in two independent experiments.

pH measurement using SNARF-1
All solutions were imaged in coated 96-well plates with sigmacote, according to a previously reported protocol by Rosen and coworkers63. Briefly, plates were first cleaned by incubation in 2% cuvette cleaner solution (30 min), followed by extensive rinsing with tap water and DDW and air-drying. Then, wells were incubated with 1 or 10 M KOH (200 μL per well) for 1 h, rinsed thoroughly with tap water and DDW, and dried. Wells were then coated with Sigmacote (100 μL per well, immediately removed), air-dried for 10 min, washed twice with isopropanol, and dried in a chemical hood. Coated plates were sealed and stored at 4°C until use. Before each experiment, the well was incubated for ~30 min with Pluronic F-127 solution (5 mg/mL in buffer without ZnCl ), washed five times with buffer, and the sample was added immediately thereafter. Final peptide concentrations were 0.5 mM and 3 mM for the Zn²⁺-dependent and Zn²⁺-free systems, respectively. SNARF-1 was dissolved in DMSO to prepare a 10 mM stock solution, which was further diluted into 1.8 mM aliquots (18% DMSO in DDW) and stored at −20 °C. All samples contained SNARF-1 at a final concentration of 20 µM (0.2% DMSO) and MeCN at a final concentration of 2.8%. 50 µl were transferred to a coated plate and incubated. Zn²⁺-dependent systems were incubated in the plate for 60 min prior to imaging, whereas Zn²⁺-free systems were incubated for 30 min. Calibration curves were constructed using buffers spanning pH 6.5–9.0 in 0.5 pH unit intervals, prepared in 10 mM Tris–HCl containing 0/0.20/0.33/0.67 mM ZnCl₂. For calibration curves measurements, five different background ROIs were selected per well in a single imaging repeat. Confocal images were acquired using a laser scanning confocal microscope (Zeiss LSM 900 inverted microscope) equipped with a 63×/1.40 oil DIC M27 objective. SNARF-1 fluorescence was excited at 488 nm, and emission was collected in two spectral windows (570–617 nm and 630–700 nm). Z-stacks were acquired with a spacing of 0.25 µm. Images were collected and processed using Zen Blue software. Images were taken at 2048×2048 pixels. For visualization and image export of 0.67 mM ZnCl , the histogram was cut and the upper limit was adjusted to 150 (white).

Image analysis and pH determination
Fluorescence intensities were extracted from the focal plane corresponding to the maximal probe signal within the relevant compartment, based on Z-stack acquisition. For each ROI, fluorescence was collected from the two emission channels and ratiometric values were calculated. For calibration curves, the ROI size was set to the average ROI size used for condensate analysis within each system. Calibration curves were fitted using a logistic function. For condensate analysis, circular ROIs with diameters slightly smaller than the corresponding droplet size were used to avoid edge effects. ROI diameter ranges [µm] were 4.69–5.95, 5.17–6.92, 5.06–6.44, 5.73–6.42, and 5.09–6.93 for R2H/E2H + 0.20 mM ZnCl , R2H/E2H + 0.33 mM ZnCl R2H/E2H + 0.67 mM ZnCl , R2H/E and R/E respectively. For analysis of the dilute phase, circular ROIs with a diameter of 3 µm were placed throughout background regions of the image, ensuring no overlap between ROIs and avoiding condensates in the current or adjacent focal planes. When condensates within the same image were present in different focal planes within the same field of view, background ROIs were selected independently in each correspond plane and averaged. The number of background ROIs per image varied due to differences in condensate density. Fluorescence ratios from all background ROIs within the same image were averaged to yield a single background value per image, which was then used for pH calculation. pH values were determined by solving the resulting equations using Wolfram Alpha.

Spectroscopic measurements
The fluorescence intensity was measured (λ = 488 nm, λ = 520-700 nm) in a 384-well black plate with a clear bottom by a Biotek H1 synergy plate reader.

FRAP CLSM analysis
FRAP experiments were performed using a Zeiss LSM 900 confocal microscope to track the fluorescence of 1% Rhodamine B as payload from 10 µM stock (in DDW). Peptide solutions were dissolved in 10 mM Tris-HCl with 0/0.20/0.33/0.67 mM ZnCl and the pH was adjusted to 7.50 ± 0.04, as described above with 0.5 mM and 3 mM final concentrations of each peptide for the Zn2+-dependent and Zn2+-free system, respectively. Condensate solutions were prepared in Eppendorf tubes with MeCN (final concentration of 2.8%), 50 µl were transferred to a coated plate and incubated for 30-60 min before the experiment. All solutions were imaged in Pluronic F-127–coated 96-well plates (5 mg/mL in buffer without ZnCl ) according to a previously reported protocol by Rosen and coworkers63, using 10M KOH. Photobleaching was performed on a circular region of interest (ROI) with a diameter of 1 μm, with a reference ROI of the same size and a background reference ROI of 1 μm. Condensates were 5.6-6.8 µm in diameter for Zn2+-dependent condensates and 5.6-6.5 µm in diameter for Zn2+-free. Five iterations of the 488 nm and 561 nm excitation lasers at 100% intensity each were applied for rhodamine B using a 40×/1.2 Imm Korr DIC M27 objective. Images were taken at 256×256 pixels. Fluorescence recovery at the bleached area was recorded and analyzed using Zen Blue 3.2 software. Photobleaching correction and recovery times were calculated using OriginPro 2025, learning edition software. Intensity data were normalized between 0% (bleach intensity) and 100% (pre-bleach intensity) using the following equation for raw data correction:
(3) I_corrected(t) = (R1(t)-R3(t)) * (R2_pre-R3_pre) / (R2(t)-R3(t))
R (t) is the FRAP region at time t, R (t) is the reference region at time t, R (t) is the background refence, R pre is pre-bleach average for the refence area (R ) and R pre is pre-bleach average for the background area (R ).

The following equation (4) used for min-max scaling (0% to 100% normalization):
(4) I_normalized(t) = (I_corrected(t)-I_min) * 100 / (I_pre-I_min)
I (t) is the calculated intensity from equation (2), I is the minimum fluorescence intensity corrected min and I denotes the pre-bleach average of the fluorescence intensity. pre

Then, the normalized data was fitted to the next equation to extract the fitting parameters:
y = a-bcx, when a is the recovery that was measured. t values of were calculated for each droplet using a,b,c extracted from the fitting, with the following equation: t = log (0.5*a / b) / c.

t values were calculated for each condensate separately, at 180 sec for Zn2+-dependent condensates and 50 sec for Zn2+-free condensates. Recovery curves represent averages of recovery plots, at 5 sec intervals for Zn2+-dependent condensates and at 1 sec intervals for Zn2+-free condensates.

CLSM microscopy imaging of coalescence
All solutions were imaged in Pluronic F-127–coated 96-well plates (5 mg/mL in buffer without ZnCl ) as described above. Peptide solutions were dissolved in 10 mM Tris-HCl with 0/0.20/0.33/0.67 mM ZnCl and the pH was adjusted to 7.50 ± 0.04, as described above with 0.5 mM and 3 mM final concentrations of each peptide for the Zn2+-dependent and Zn2+-free systems, respectively. All samples were prepared directly in the well and contained MeCN at final concentration of 2.8%. Zn2+-dependent systems were incubated for ~30 min in the plate and Zn2+-free were imaged almost immediately after preparation. Images were taken using a laser scanning confocal microscope (Zeiss LSM 900 inverted microscope), 40×/1.2 Imm Korr DIC M27 objective. Images were collected and processed using Zen Blue software. For visualization and image export, the histogram upper limit was adjusted to 150 (white). Videos were exported at different frame rates to reduce file size, as detailed here: R2H/E2H + 0.20 mM ZnCl 47.4 fps; R2H/E2H + 0.33 mM ZnCl 15.80 fps; R2H/E2H + 0.67 mM ZnCl 15.80 fps; R2H/E 31.60 fps; R/E 31.60 fps. The experiment was performed once.

STEM-EDS
Samples were prepared as previously described. 2 μl of the sample solution were applied to a FCF400-Cu grid (FORMVAR/Carbon Film, 400 mesh copper) and incubated for 2 min. Excess solution was removed by blotting the grid with a filter paper, then the grid was left to air-dry for 10 min. Imaging and elemental mapping were performed on a probe‑corrected Spectra 200 (S)TEM (Thermo Fisher Scientific) equipped with an X‑type cold field‑emission gun and a Super‑X EDS system (Thermo Fisher Scientific). Operating conditions: 80 kV accelerating voltage, probe current ~95 pA, probe collection semi‑angle ~30°, camera length 98 mm, column pressure ~5 × 10−6 Pa. Images were acquired using segmented bright‑/dark‑field detectors (Panther, 8 segments) and a HAADF detector (Fischione Instruments). Data were acquired and processed in Velox 3.15 (Thermo Fisher Scientific) . For elemental mapping, zinc used as marker; line‑scan intensities were normalized as (I x − I min )/(I max − I min ). Analysis performed on 3 condensates of each system and the data represent one condensate.

EE analysis
Calibration curves:
Stock solution of 4-MU (36 mM) was prepared in acetonitrile. It was diluted with acetonitrile to vary concentration (14.4, 11.52, 8.64, 5.76 and 2.88 mM). 1 μl of each 4-MU concentration was added to a 384 well plate with 35 μl of 10 mM Tris buffer solution to final 4-MU concentrations of 0.4, 0.32, 0.24, 0.16 and 0.08 mM, respectively. Absorbance spectra of quadruplicates measured at range of 300-400 nm in a 384 well black plate by Biotek H1 synergy plate reader (purchased from Lumitron, Israel). The calibration curves were calculated at 322 nm.

Zn2+-dependent systems using spectroscopic measurements
solutions of 1.5 mM E2H and R2H peptides were prepared in 10mM Tris with 0.2, 0.33 and 0.67 mM ZnCl at pH 7.5±0.04. A solution of a final concentration of 0.5 mM of each peptide with 1 mM of 4-MU were made at volume of 144 μl in a 1.5ml Eppendorf tube. For control samples acetonitrile was added instead of 4-MU. After 10 min of incubation, the samples were centrifuged at 1,500 × g for 7 minutes. A volume of 120 μl from the supernatant was collected and diluted 4 times with 10 mM Tris buffer. The absorbance of triplicates of 36 μl of the diluted supernatant was measured at λ = 322 nm in a 384 well black plate by Biotek H1 synergy plate reader (purchased from Lumitron, Israel). All experiments were performed in triplicate. The concentration of the supernatant solutions is determined by the calibration curves. %EE was calculated using Eq. 1.

Zn2+-free system using CLSM
All solutions were imaged in Pluronic F-127–coated 96-well plates (5 mg/mL in buffer without ZnCl ) as described for FRAP. Peptide stock solutions (9 mM) were dissolved in 10 mM Tris-HCl and the pH was adjusted to 7.50 ± 0.04. Condensate solutions were prepared in Eppendorf tubes with 0.2 mM (2.8% MeCN) final concentration of 4-MU and 3 mM of each peptide, 50 µl were transferred to a coated plate and incubated for 30 min before the experiment. z-stack imaging with spacing of 0.2 µm were taken. Images show the z-stacking middle section and were taken using a Zeiss LSM 900 CLSM, 40×/1.2 Imm Korr DIC M27 objective. Images were taken at 1024×1024 pixels. Fluorescence images were taken using 405 nm laser. Images were collected and analyzed using Zen Blue software. Condensates were 2.5-4.2 µm in diameter. Each experiment repeated itself twice.

CLSM imaging of 4-MU-Ac hydrolysis
All solutions were imaged in Pluronic F-127–coated 96-well plates (5 mg/mL in buffer without ZnCl ) as described for FRAP (without incubation of samples). Reactions were prepared as detailed above for reaction kinetics analysis, 4-MU-Ac at 1.1 mM final concentration was added directly in the well. Z-stack images were taken immediately after adding the substrate solution and every 30 minutes up to 60 minutes. z-stack imaging with spacing of 0.2 µm were taken in each time point and each experiment repeated itself three times. Images show the z-stacking middle section and were taken using a Zeiss LSM 900 CLSM, 40×/1.2 Imm Korr DIC M27 objective. Images were taken at 1024×1024 pixels. Fluorescence images were taken using 405 nm laser. Images were collected using Zen Blue software. Each experiment repeated itself three times.

Crystallization analysis
Stock solution of 4-MU-Ac was prepared in MeCN, final substrate concentration was either 1.1 mM or 0.7 mM. Stock solutions of R2H and E were prepared at 9 mM in 10 mM Tris-HCl and the pH of each solution was adjusted to 7.50 ± 0.04. Samples were formed by adding buffer, R2H and E (for condensates) to a final concentration of 3 mM of each peptide. Substrate was added lastly from the appropriate stock by pipetting into the well. Bright field images were taken for three samples by fluorescence microscope (Olympus), x10/0.25 NA Plan Apochromat objective (for soluble R2H with 0.7 mM 4-MU-Ac) and by x40/0.95 NA Plan Extended Apochromat objective (for R2H/E condensates and soluble R2H with 1.1 mM 4-MU-Ac). All samples were loaded on a 96 well Black Glass bottom plate. Images were collected and processed using CellSens Dimension software.

Soluble R2H with 0.7 mM 4-MU-Ac
Images were collected over a time lapse of 15 min. Each well was scanned in a 3 by 3 array with spacing of 86 µm between the rows and columns and a z-stack with spacing of 20 µm at a total range of 200 µm. Each experiment repeated itself three times.

R2H/E condensates and soluble R2H with 1.1 mM 4-MU-Ac
Images were collected manually by scanning the well at different locations and z-stacks for 15 min. Each experiment repeated itself three times.

Dilute phase kinetics
Peptide stock solutions were prepared at final concentration of 3 mM of each peptide in 10 mM Tris-HCl and the pH of each solution was adjusted to 7.50 ± 0.04. Condensate were prepared as described above. Samples were centrifuged at 17,700 RCF for 4 h at 10 °C. The supernatants were collected and imaged immediately as described above. Kinetics measurements were conducted as described above. Dilute phase kinetics were calculated using a calibration curve constructed in buffer under identical experimental conditions.

Mass spectrometry analysis
Samples were prepared following the same protocol as the kinetic assays, with a final substrate concentration of 0.7 mM. After ~30 min incubation at room temperature, an equal volume of acetonitrile was added to disrupt the condensates, and samples were collected for further analysis. The analysis was performed once for representative samples under each condition to confirm product identity. High-resolution mass spectrometry (HRMS) analyses were performed on a Waters G2 XS QTOF instrument using both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in positive ion mode. Samples were analyzed by direct injection. The molecular ion corresponding to the protonated species [M+H] + was measured in APCI mode. The calculated exact mass for [M+H] + of C₁₀H₉O₃ is m/z=177.0552. The experimentally determined masses were as follows: R2H/E2H + 0.20 mM ZnCl , m/z=177.0549 (Δ = 0.3 mDa, 1.7 ppm); R2H/E2H + 0.33 mM ZnCl , m/z 177.0553 (Δ = 0.1 mDa, 0.6 ppm); and R2H/E2H + 0.67 mM ZnCl , m/z=177.0552 (Δ = 0.0 mDa, 0.0 ppm) ; R2H/E, m/z=177.0551 (Δ = 0.1 mDa, 0.6 ppm); R1H/E, m/z =177.0547 (Δ = 0.5 mDa, 2.8 ppm); R/E, m/z=177.0549 (Δ = 0.3 mDa, 1.7 ppm).

DLS analysis
Zn+2-mediated condensates were prepared as follows: Stock solutions of positively charged peptide (solution A) and negatively charged peptide (solution B) were prepared at 1.5 mM in a 0.67 mM ZnCl solution in 10 mM Tris-HCl and the pH of each solution was adjusted to 7.50 ± 0.04. Samples were formed by adding buffer, solution A, solution B, and MeCN (final concentration of 2.8%) at final concentration of 0.5 mM for R2H/E2H.

Zn+2-free condensates were prepared as follows: Stock solutions of solution A and solution B were prepared at 9 mM in 10 mM Tris-HCl and the pH of each solution was adjusted to 7.50 ± 0.04. Samples were formed by adding buffer, solution A, solution B and MeCN (final concentration of 2.8%), at final concentrations of 3 mM for R/E, R1H/E and R2H/E.

Each sample was placed in ZEN0040 cuvettes and were measured by Malvern - Zetasizer nano Z (purchased from Dr. Golik). The diameter of the condensates was calculated as the average of 14 repeats. Each experiment was conducted in triplicates.

CD
Peptide solutions for CD were prepared at concentration of 0.5 mM in 10 mM Tris buffer solution with 0.2, 0.33, 0.67 mM of ZnCl and with no ZnCl (in samples of mixtures of peptides, each peptide was at concentration of 0.5 mM). pH was adjusted to 7.5 ±0.04. The solutions were placed in a 0.2 mm path length quartz cuvette at 25 °C, and the range of 190–260 nm was recorded on a Chirascan spectrometer. Background (buffer with suitable ZnCl concentration) was subtracted from the CD spectra.

Trp Emission analysis
Samples of R, R2H, E and E2H peptides were prepared at final concentration of 0.5mM with and without 0.67 mM ZnCl in 10 mM Tris-HCl and the pH of each solution was adjusted to 7.50 ± 0.04. The fluorescence intensity was measured (λ = 280 nm, λ = 300-500 nm) in a 384-well black plate with a clear bottom by a Biotek H1 synergy plate reader.

Microscopy analysis of K2H/E, K/E
Stock solutions were prepared at 20 mM (final concentration of 10 mM of each peptide) in 10 mM Tris-HCl and the pH of each solution was adjusted to 7.50 ± 0.04. Samples were prepared by mixing K2H or K solution with E solution at 1:1 ratio. Samples were imaged as described above for the phase diagrams.

TEM analysis
R2H sample was prepared at a final concentration of 3 mM, in 10 mM Tris-HCl buffer with 2.8% MeCN, pH 7.50 ± 0.04. An aliquot of 5 μL of the sample solution was applied to a FCF400-Cu grid (FORMVAR/Carbon Film, 400 mesh copper) and incubated for 2 min. Excess solution was removed by blotting the grid with a piece of filter paper, followed by staining with 5 μL of 50% (v/v) uranyless for 1 min. After blotting excess stain solution, the grid was left to air-dry. Buffer sample was prepared the same way. The negatively stained sample was imaged by a Talos F200i (S)TEM operated at 80 kV. Images were recorded using a Ceta-M 4K×4K detector.

MD simulations
The initial structures of the peptides used in the simulation (including R, R1H, R2H, E2H) were generated using ESMFold 64. Subsequently, the structure of each single sequence was sampled using simulated annealing. Each polypeptide was solvated in a cubic aqueous box, ensuring a minimum distance of 15 Å between the peptide and the periodic boundary. The system then underwent energy minimization to remove potential steric clashes. An annealing step was then performed by heating the system to 2000 K for 5 ns, followed by a cooling and equilibration phase at 300 K for an additional 15 ns. To extensively sample the conformational landscape, 40 independent, parallel simulations were carried out for each polypeptide. To construct the multi-peptide systems, 20 peptide conformations, obtained from the preceding simulated annealing trajectories, were randomly placed within a cubic periodic box with dimensions of 12.7 × 12.7 × 12.7 nm3. This arrangement resulted in a concentration of approximately 19 mM for each polypeptide species. For simulation systems containing only the R2H sequence, Cl- ions were added to neutralize the charge. In systems designed to investigate the effects of Zn²⁺, 150 mM ZnCl was added. The entire system included approximately 68,000 water molecules and 210,000 atoms. Details of the simulated systems is listed in Supplementary Table 10.

The simulations were performed using the CHAR MM36m force field 65 and the TIP3P water model 66. All simulations were performed using GROMACS 2022 67. Non-bonded van der Waals interactions were calculated using a cutoff of 1.2 nm, with a force-switching function applied between 1.0 and 1.2 nm to smoothly taper the potential to zero. Long-range electrostatic interactions were treated with the Particle Mesh Ewald (PME) method, employing a real-space cutoff of 1.2 nm. The system was energy minimized to remove bad contacts. Then the system was heated to 300 K at a constant volume using velocity-rescaling thermostat. Production simulations were then continued in the NPT ensemble at 1 atm pressure and 300 K for 400 ns. Temperature and pressure were controlled by the Nosé-Hoover thermostat68,69 and the Parrinello-Rahman barostat70, with coupling time constants of 1.0 ps and 5.0 ps, respectively. Bonds involving hydrogen atoms were constrained by the LINCS algorithm 71, and a time step of 2 fs was used. System trajectories were saved every 100 ps for subsequent analysis. The first 200 ns of the production simulation were treated as equilibration and discarded from subsequent statistical analyses. We carried out three replicate MD simulations for each system and obtained consistent results. To investigate the impact of pH variations, we modulated the protonation states of histidine residues within the R2H/E system to varying degrees. Snapshots at 400 ns was extracted from replicate simulations of the fully neutral system to serve as the initial structure for these new simulations. Subsequently, 1/3 and 2/3 of the R2H peptides were randomly selected, and one histidine residue on each selected chain was protonated. Following energy minimization, 100 ns simulations were conducted under identical simulation parameters. Data from the final 10 ns of the trajectories were used to analyze the system properties under different protonation states.

UCSF ChimeraX 72 and VMD 62 were used to analyze the MDS simulation trajectories.

Ab initio calculations
In the active site model of the system containing Zn2 +, three His residues participate in coordination. Two His residues retain their side chains and Cα atoms, while one His additionally retains the carboxylate group at its C-terminal backbone. The three His residues coordinate with Zn²⁺ through their N atoms. The C atoms of the His are frozen to mimic the constraints resulting from interactions within the condensate. When comes to the Zn2+-free catalytic center, structures sampled from MD simulations was used to build the initial structure, and the C atoms of the two His are also frozen to mimic the condensate environment.

All geometry optimizations and frequency analysis were performed using M06-2X73/def-TZVP74 with the PCM solvation model75. For electronic energies calculations, the larger def2-TZVP basis set76 was used along with the SMD solvation model77. For systems containing Zn2+, LANL2DZ78 was used to describe Zn2+ in geometry optimizations, while SDD79 was used in electronic energies calculation. Thermodynamic corrections from frequency analysis were incorporated into the solution-phase single-point energy to obtain Gibbs free energy, which were then used in the mechanistic discussion. All ab initio calculations were conducted using the Gaussian16 program60.

Statistics and Reproducibility
For kinetics measurements, for each dataset, the mean of all measurements within a given condition was calculated, and the percentage deviation of each value from the mean was determined. All available measurements were included, even those from days with fewer replicates (minimum two replicates). To ensure consistency and accuracy in data analysis, exclusion criteria were determined for calibration curves and kinetic measurements based on the amount of data available and the range of raw data values. Outliers falling outside the range of more than 25% above or below the mean for kinetics data, or more than 30% above or below the mean for calibration curves, were excluded from the statistics. Separate cutoff ranges were selected to account for the differing variability in these datasets, with calibration curves exhibiting inherently higher variability. This approach ensured sufficient data retention to construct reliable calibration curves. Background measurements, which have very low fluorescence intensities and are inherently noisy, were visually inspected using graph representations, using only the first measurement. All calculations and exclusions were systematically documented to ensure reproducibility and transparency. Detailed experimental replicates are detailed in Supplementary Table 11. p-values are reported in the figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This paper demonstrated that: condensates formed by minimalistic histidine-containing peptides can catalyze ester hydrolysis via two pathways: a Zn²⁺-dependent mechanism activating bound water and a metal-free mechanism driven by LBHB. The concept is of highly interested and the discovery is exciting. However, the authors want to address the following technical considerations before this can be published.\n1. This work suffers from a fundamental flaw, namely the overlooking of the effect of pH on hydrolysis reactions. It is well known that pH affects hydrolysis reactions. Recently, more and more studies have shown that phase separation can create a distinct pH within condensates (see, doi.org/10.1101/2024.05.23.595321). The mechanism underlying this distinct pH environment is the partitioning of ions (including protons) during LLPS. In this study, the authors tested (1) condensates made of different peptides and (2) condensates formed in the presence of Zn²⁺ or other ions , both conditions that can create distinct intra-condensate pH. Therefore, the authors should first demonstrate that\n(1) condensates made of different peptides possess the same (or at least similar) pH;\n(2) condensates formed at different Zn²⁺ concentrations possess the same (or at least similar) pH.\nIntra-condensate pH can be measured with fluorescent probes.\n(3) study how distinct pH can modulate the reactions.\nThese experiments will provide alternative explanations to the current observations.\n2. If, as is very likely, condensates made of different peptides possess different pH values, how do the authors explain the mechanisms underlying the observed hydrolytic activities?\n3. The authors showed that condensates formed in the presence of Zn²⁺ become more compact, which in turn lowers their hydrolytic capability. However, more compact condensates may also be more hydrophobic inside, which would facilitate the partitioning of low-polarity molecules such as pNPA or 4-methylumbelliferyl acetate. Therefore, the authors should directly demonstrate the limited partitioning of 4-methylumbelliferyl acetate, for example by directly using 4-MU.\n4. Using rhodamine B to probe partitioning and diffusion in this coacervate system can be problematic. Rhodamine B is a charged molecule, so its partitioning and diffusion can be affected by electrostatic interactions with the charged peptides. Because the hydrolysis substrates used in this article are non-charged, the authors should replace rhodamine B with a neutral fluorescent molecule.\n5. The authors investigated the mechanism underlying the Zn²⁺-independent hydrolytic activity of the coacervates and attributed it to LBHB formation. However, evidence for LBHBs within the Zn²⁺-containing condensates is lacking. Since Zn²⁺ makes the condensates more compact, it could, in principle, promote LBHB formation and thus enhance the reaction — which would contradict the authors’ conclusion that Zn²⁺ inhibits the hydrolytic capability of the coacervates. The authors should clarify this point.\n6. For experimental rigor, please provide mass spectrometry data verifying the occurrence of hydrolysis reactions.", "reviewer_2": "The revised manuscript by Massarano et al. has been significantly improved compared to the original version in terms of both novelty and experimental rigor. First, the authors have reinforced the discussion part to clearly address the conceptual novelty that their work has demonstrated, including intrinsic catalytic properties and Zn-dependent switching between two distinct mechanisms of catalysis. (which is now also conveyed in the revised title). Additionally, the authors have included significant amount of rigorous kinetic studies, in addition to the FRAP and DLS experiments that were originally missing, to demonstrate the sequence-based control over the condensate catalytic and material characteristics.\nTherefore, the authors have answered all of my points and concerns provided in the first round of review. I believe that the manuscript is now ready for the publication, and thus recommend it to be accepted. I believe that this work will provide a novel addition to the research field of LLPS, and improve our understanding of how condensate properties could be precisely tuned in LLPS-based systems.", "reviewer_4": "The manuscript by Lampel and co-workers describes the formation of LLPS peptide condensates capable of catalysing ester hydrolysis through either Zn²⁺-dependent or Zn²⁺-independent mechanisms. The authors combine microscopy, kinetic measurements, and computational studies to explore how histidine residues and metal coordination contribute to catalysis. \nOverall, the manuscript lacks a clear hypothesis-driven design, quantitative experimental validation of the proposed mechanisms, and the level of mechanistic generality expected for the journal.\nConsidering the level of novelty of the topic and the scope of the study, I do not believe that the manuscript is suitable for publication in Nature Communications, which addresses a broader multidisciplinary audience. A more specialised chemistry journal focusing on peptide-based catalysis or supramolecular systems would likely be a more appropriate venue.\n• The manuscript focuses on peptide LLPS systems and their catalytic activity toward ester hydrolysis. Although this topic remains of chemical interest, it does not represent a particularly timely or conceptually novel advance for the Nature portfolio. Several studies have already reported catalytic coacervates and peptide condensates employing similar model reactions. The present work refines rather than expands the existing framework and therefore does not meet the broad conceptual impact expected for this journal.\n• The rationale behind the experimental sequence requires clearer justification. The authors initially state that Zn²⁺ promotes LLPS and contributes to catalysis, but the data indicate that higher Zn²⁺ concentrations inhibit activity. This outcome is intriguing yet undermines the central design logic. It remains unclear why the system was optimised around Zn²⁺ if Zn²⁺ coordination ultimately suppresses reactivity. The authors should explicitly address whether the Zn²⁺-free system was conceived as a control or as an alternative catalytic regime.\n• Some figure references are inconsistent or incomplete (e.g., “Fig. 1b–g” does not correspond to any figure panels). Furthermore, key datasets are represented only as heat maps or schematic diagrams, with no access to the raw numerical data. Providing the quantitative values underlying the phase diagrams and kinetic analyses would be essential for reproducibility and for assessing data quality.\n• The level of characterisation is insufficient to support the claim that bona fide LLPS condensates are formed. Bright-field microscopy, DLS, and FRAP provide morphological information but do not establish the molecular organisation or Zn²⁺ coordination environment. Complementary spectroscopic data (e.g., circular dichroism, FTIR, or NMR) would be required to confirm peptide structure and metal binding. Without these, it remains uncertain whether the observed droplets are genuine dynamic condensates or partially aggregated assemblies.\n• The kinetic studies rely on a single substrate (4-MU-acetate), and the comparison to literature values obtained with other substrates (e.g., p-nitrophenyl acetate) is not meaningful. The mechanistic claims rely entirely on computational models, without experimental validation through pH-rate profiles, isotope effects, or metal titrations. These additional experiments would be essential to establish whether the observed catalysis indeed proceeds through the proposed Zn²⁺-dependent and Zn²⁺-independent pathways.\n• The manuscript would benefit from a clearer structure separating experimental results from interpretation. The text is occasionally difficult to follow, and figure descriptions are fragmented between the main text and the Supplementary Information. The inclusion of peptide sequences or schematic representations in the main figures (currently confined to Table S1) would greatly improve readability.\nI also evaluated the responses to Reviewer 3's comments. I can confirm that the comments raised by Reviewer 3 have been satisfactorily addressed."}, "author_response": "Point-by-point Responses to Reviewers comments:\nReviewer #1\nThis paper demonstrated that: condensates formed by minimalistic histidine-\ncontaining peptides can catalyze ester hydrolysis via two pathways: a Zn²⁺-dependent\nmechanism activating bound water and a metal-free mechanism driven by LBHB. The\nconcept is of highly interested and the discovery is exciting. However, the authors want\nto address the following technical considerations before this can be published.\n1. This work suffers from a fundamental flaw, namely the overlooking of the effect of pH\non hydrolysis reactions. It is well known that pH affects hydrolysis reactions. Recently,\nmore and more studies have shown that phase separation can create a distinct pH\nwithin condensates (see, doi.org/10.1101/2024.05.23.595321). The mechanism\nunderlying this distinct pH environment is the partitioning of ions (including protons)\nduring LLPS. In this study, the authors tested (1) condensates made of different\npeptides and (2) condensates formed in the presence of Zn²⁺ or other ions , both\nconditions that can create distinct intra-condensate pH. Therefore, the authors should\nfirst demonstrate that\n(1) condensates made of different peptides possess the same (or at least similar) pH;\n(2) condensates formed at different Zn²⁺ concentrations possess the same (or at least\nsimilar) pH.\nIntra-condensate pH can be measured with fluorescent probes.\n(3) study how distinct pH can modulate the reactions.\nThese experiments will provide alternative explanations to the current observations.\nResponse: We thank the reviewer for the overall positive feedback and for the\nconstructive and important comment.\nWe agree that pH is a critical factor for hydrolysis reactions and that measuring the pH in\nthe dilute vs dense phase of the different condensate systems in this study can provide\nnew insights.\nTo address the comment, we have followed the study mentioned by the reviewer\n(Knowles and coworkers bioRxiv) as well as studies by Arosio (Nat. Commun. 2025) and\nChilkoti and coworkers (Chem 9, no. 6 (2023): 1594-1609) and performed emission\nspectroscopy and CLSM analysis using the pH probe SNARF-1. For this, we first analyzed\nhow buffer pH affects the ratio between the two emission maxima of the probe, at\n =580 nm and  =640. This results in presented in Figure S8 of the revised SI.\nem em\nNext, we used CLSM to create calibration curves for each buffer by measuring the\nfluorescence intensity of SNARF-1 using two channels,  =580 nm and  =640. The\nem em\nvarious calibration curves included Tris buffer without ZnCl and with 0.2, 0.33, and 0.67\n2\nmM of ZnCl . We next analyzed the I ratio of R2H/E2H condensates formed with\n2 580nm/640nm\nZnCl at varying concentrations. We found that the dense phase of all three condensates\n2\nis more basic than the dilute phase, where the dense-phase pH of condensates formed\nwith the lower ZnCl concentration (0.2 mM and 0.33 mM) found to be >9 and more basic\n2\ncompared to that with the highest ZnCl (0.67 mM). We attribute the difference in dense-\n2\nphase pH at low and high Zn2+ concentration to Zn²⁺-His coordination. At lower Zn²⁺,\nfewer His residues are coordinated, increasing the effective basicity of the dense phase.\nAt higher Zn²⁺, extensive Zn²⁺-His coordination reduces the availability and basicity of\nfree His residues, resulting in a lower dense-phase pH. The elevated basicity observed at\nlow Zn²⁺ is expected to promote the hydrolytic reaction. For the Zn²⁺- free system, both\nthe dilute and the dense phases of R2H/E and R/E were found to be basic, and no\nsignificant differences between the systems. To test how basicity affects the reaction, we\nanalyzed the kinetics of hydrolysis at pH 7.5 and 9.0 (presented in Fig. S10 and S15).\nIndeed, the basic pH accelerated the kinetics of the reaction.\nFor two systems, R2H/E2H at 0.33 mM Zn²⁺ and R2H/E we were unable to determine the\nexact dense-phase pH value as the 580/640 nm intensity ratio of these systems lies\noutside the range of the fitting model equation applied for the calibration curves. Yet the\nresults indicate that these pH values are > 9.0.\nThe pH analysis was included in Fig. 2 and Fig. 5 and the values are presented in Table S6\nof the SI. The following text was added to the MS (page 8 line 5): “To further elucidate how\nZn²⁺ concentration influences the catalytic activity of the phase-separated systems, we\nmeasured the pH of both the dilute and dense phases using the ratiometric pH probe\nSNARF-1 and by confocal laser scanning microscopy (CLSM)47,53,54. The emission ratio of\nSNARF-1 at λ = 580 and 640 nm varies as a function of pH (Fig. S8). Calibration curves\nem\nwere obtained by measuring the I ratio in buffers of defined pH containing varying\n580/640\nZn²⁺ concentrations, enabling quantitative determination of pH in each phase (Fig. 2f–g).\nThe pH of the dilute phase was similar across all systems, with 7.7 ± 0.11 and 7.6 ± 0.40\nfor 0.2 mM and 0.67 mM Zn²⁺, respectively. In contrast, the dense-phase pH was strongly\ndependent on Zn²⁺ concentration, decreasing from a highly basic value of 10.8 ± 0.60 at\n0.2 mM Zn²⁺ to 8.5 ± 0.10 at 0.67 mM Zn²⁺ (Fig. 2f–g, Table S6).”\nPage 9 line 13: “The difference in dense-phase pH at varying Zn2+ concentration can be\nattributed to Zn²⁺-His coordination. At lower Zn²⁺ concentrations, fewer His residues are\ncoordinated, increasing the effective basicity of the dense phase. At higher Zn²⁺\nconcentrations, extensive Zn²⁺-His coordination reduces the availability and basicity of\nfree His residues, resulting in a lower dense-phase pH. The elevated basicity observed at\nlow Zn²⁺ is expected to promote the hydrolytic reaction, consistent with enhanced\nreaction rate measured in bulk buffer under basic conditions (Fig. S10) and provides a\nmechanistic basis for the highest catalytic activity observed at 0.2 mM Zn²⁺.\nPage 15 line 21: “To shed light on the catalytic capacity of the condensates, we measured\nthe pH of the dense and dilute phases using the ratiometric probe SNARF-1. Both phases\nin the R2H/E and R/E systems were found to be basic, with dilute-phase pH values of 8.0\n± 0.1 and 8.4 ± 0.3, respectively, and no significant differences between the systems (Fig.\n5d–e, Table S6). The basic environment in both phases is expected to promote the\nhydrolytic reaction, consistent with the enhanced catalytic activity observed in bulk\nbuffer under basic conditions (Fig. S15).”\nThese new insights are also reflected in the revised Discussion and Abstract.\n2. If, as is very likely, condensates made of different peptides possess different pH\nvalues, how do the authors explain the mechanisms underlying the observed hydrolytic\nactivities?\nResponse: The overall basic pH found in all systems is expected to increase the\nefficiency of the reaction as detailed in the response to the previous comment (and as\npresented in Fig. S10 and Fig. S15). As mentioned above, we did not find a significant\ndifference between the dilute and dense-pH values of different peptides (R2H/E vs. R/E).\nAs histidine is a titratable amino acid, its protonation behavior offers valuable insights\ninto the unique local acid-base variations characteristic of the droplet. We modulated\nthe protonation states of histidine residues within the R2H/E system to varying degrees\nin MD simulations. Considering that the pKa for the complete deprotonation of His to\nform its anionic form is approximately 13, we only consider its neutral and cationic forms.\nThe detailed information can be found in Table S9.\nAs shown in Figure S22, we compared the collapse degree and the pairwise histidines’\nCα-Cα distance distribution across neutral and partially protonated systems. The results\ndemonstrate that histidine state did not significantly alter the LLPS properties of the\npeptide droplet (degree of collapse). Furthermore, the Cα distance distribution remained\nlargely unaffected, suggesting that the structural prerequisites for LBHB formation are\nmaintained. These results suggest that while the local acid-base environment within the\ndroplet may differ from that of the bulk phase, our proposed molecular mechanism\nremains relatively robust to pH variations. This stability might be attributed to histidine\npartially acting as a buffer agent, effectively maintaining the internal structural integrity\nof the LLPS under varying solution conditions.\nThe results of the simulation are presented in Fig. S22 and Table S9 and the connection\nto the catalytic mechanism was clarified in the text as follows (page 18 line 5): “Lastly, we\nmodulated the protonation states of His residues within the R2H/E system to varying\ndegrees in MD simulations (Table S9). Considering that the pKa for the complete\ndeprotonation of His to form its anionic form is approximately 13, we only consider its\nneutral and cationic forms. We compared the collapse degree and the pairwise His’ Cα-\nCα distance distribution across neutral and partially protonated systems (Fig. S22). The\nresults demonstrate that His state does not significantly alter the LLPS properties of the\npeptide droplet (degree of collapse). Furthermore, the Cα distance distribution remained\nlargely unaffected, suggesting that the structural prerequisites for LBHB formation are\nmaintained. These results suggest that while the local acid-base environment within the\ndroplet may differ from that of the bulk phase, our proposed molecular mechanism\nremains relatively robust to pH variations. This stability might be attributed to histidine\npartially acting as a buffer agent, effectively maintaining the internal structural integrity\nof the LLPS under varying solution conditions.”\n3. The authors showed that condensates formed in the presence of Zn²⁺ become more\ncompact, which in turn lowers their hydrolytic capability. However, more compact\ncondensates may also be more hydrophobic inside, which would facilitate the\npartitioning of low-polarity molecules such as pNPA or 4-methylumbelliferyl acetate.\nTherefore, the authors should directly demonstrate the limited partitioning of 4-\nmethylumbelliferyl acetate, for example by directly using 4-MU.\nResponse: We thank the reviewer for the comment. To directly address this point, we\nquantified the encapsulation efficiency (EE) of the 4-MU product in R2H/E2H\ncondensates formed at varying ZnCl₂ concentrations by measuring the product\nconcentration remaining in the dilute phase using absorbance spectroscopy with a\ncalibrated standard. Contrary to the expectation that increased compaction would\nenhance partitioning of low-polarity molecules, we observe an inverse correlation\nbetween Zn²⁺ concentration and 4-MU recruitment. Specifically, condensates formed at\nlower Zn²⁺ concentrations (0.2 and 0.33 mM) exhibit significantly higher 4-MU EE\ncompared to those formed at 0.67 mM Zn²⁺ (Fig. S9).\nWe attribute this trend to Zn²⁺-mediated crosslinking within the dense phase at higher\nZn²⁺ concentrations, which reduces internal diffusivity and might limit accessible free\nvolume and porosity. Thus, although higher Zn²⁺ leads to more compact condensates,\nthis compaction does not promote product partitioning; instead, it restricts molecular\nuptake and contributes to the reduced catalytic performance observed under these\nconditions.\nThe results of the EE analysis are presented in Fig. S9 of the revised SI. The following text\nwas included in the revised MS (page 9 line 10): “The partitioning of the 4-MU product was\nweakest in condensats formed at 0.67 mM Zn²⁺ (Fig. S9), likely due to the highly\ncrosslinked dense-phase network at this Zn²⁺ concentration, which reduces accessibility\nand limits internal porosity.”\n4. Using rhodamine B to probe partitioning and diffusion in this coacervate system can\nbe problematic. Rhodamine B is a charged molecule, so its partitioning and diffusion\ncan be affected by electrostatic interactions with the charged peptides. Because the\nhydrolysis substrates used in this article are non-charged, the authors should replace\nrhodamine B with a neutral fluorescent molecule.\nResponse: We agree with the reviewer that the ionizable groups of rhodamine B may\npromote electrostatic interactions with the charged peptides and thus influence its\ndiffusion within the dense phase. Nevertheless, rhodamine B is widely used in the LLPS\nliterature for FRAP measurements and is predominantly zwitterionic at neutral pH,\ncontaining both positive and negative charges that may engage in opposing (attractive\nand repulsive) interactions. More broadly, most commonly used FRAP probes in\nbiomolecular condensates, including GFP and its derivatives, also carry net charge.\nTo directly address the reviewer’s concern, we performed additional FRAP experiments\nusing two neutral fluorescent dyes: NBD-hexylamine and BODIPY. As shown in Fig. 1a,\nboth dyes exhibited extremely weak fluorescence in the dense phase and pronounced\ninhomogeneous distribution across the sample, particularly for BODIPY. We attribute this\nbehavior to non-specific adsorption to the Pluronic-coated wells and limited partitioning\nwithin the condensates.\nDespite these limitations, FRAP recovery curves were obtained and the corresponding t₁₂\n/\nvalues were extracted (Fig. 1b). While the absolute t₁₂ values for both neutral dyes are\n/\nsignificantly lower than those obtained with rhodamine B, the overall trend is preserved.\nIncreasing the Zn²⁺ concentration from 0.33 mM to 0.67 mM results in a 2.3-fold and 3-\nfold increase in t₁₂ for NBD-hexylamine and BODIPY, respectively, consistent with the\n/\ntrend observed using rhodamine B.\nWe therefore conclude that the higher absolute t₁₂ values obtained with rhodamine B\n/\nlikely reflect stronger dye–peptide interactions in the dense phase. However, due to the\nextremely weak signal and non-specific distribution of the neutral dyes, rhodamine B\nprovides more reliable FRAP data in this system. To explicitly acknowledge this limitation,\nwe have added the following text to the revised manuscript (page 8, line 19): “We note\nthat the absolute diffusivity values obtained using rhodamine B may be influenced by\nelectrostatic dye–peptide interactions.”\nFig. 1. a. Representative CLSM images of R2H/E2H condensates formed at 0.33 and 0.67 mM\nZnCl₂ and loaded with rhodamine B (0.1 µM, dissolved in DDW), NBD-hexylamine (10 µM,\ndissolved in MeCN), and BODIPY (1.5 µM, dissolved in MeCN). Scale bars = 2 µm. b. t1/2 values\nobtained based on recovery plots of FRAP analysis of R2H/E2H condensates at 0.33 and 0.67 mM\nZnCl₂. Data are presented as mean ± STD, number of analyzed droplets (n) is indicated.\n5. The authors investigated the mechanism underlying the Zn²⁺-independent hydrolytic\nactivity of the coacervates and attributed it to LBHB formation. However, evidence for\nLBHBs within the Zn²⁺-containing condensates is lacking. Since Zn²⁺ makes the\ncondensates more compact, it could, in principle, promote LBHB formation and thus\nenhance the reaction — which would contradict the authors’ conclusion that Zn²⁺\ninhibits the hydrolytic capability of the coacervates. The authors should clarify this\npoint.\nResponse: While Zn²⁺ binding does increase the overall density of the condensate, the\nspecific interaction between Zn²⁺ and histidine residues draws histidines away from each\nother, reducing the likelihood of the close proximity and optimal orientation required for\nefficient LBHB formation between histidines. Therefore, the mechanism promoting\ncompactness (Zn²⁺-His coordination) and the mechanism required for LBHB formation\n(His-His interactions) are applicable under different environmental conditions. This\ncompetition between Zn²⁺-histidine coordination and histidine-histidine interactions\nclarifies the apparent contradiction raised by the reviewer. It explains why the presence\nof Zn²⁺, despite leading to a more compact structure, results in diminished hydrolytic\nactivity, consistent with our conclusion that Zn²⁺ inhibits the LBHB-dependent pathway.\n6. For experimental rigor, please provide mass spectrometry data verifying the\noccurrence of hydrolysis reactions.\nResponse: We thank the reviewer for the comment. To verify the occurrence of ester\nhydrolysis, we analyzed reaction mixtures from all catalytic systems at t=30 min using\nhigh-resolution mass spectrometry (HR-MS). In all cases, we detected a signal\ncorresponding to the expected hydrolysis product (m/z≈177.06), confirming product\nformation. Notably, the same ion was also observed in the substrate-only control, which\nwe attribute to partial hydrolysis occurring during the MS ionization process, as well as to\nthe higher ionization efficiency of the product relative to the ester substrate.\nWhile HR-MS therefore confirms the identity of the reaction product, it does not provide\nquantitative information on reaction extent. Consequently, quantitative assessment of\ncatalytic activity was performed using time-resolved fluorescence spectroscopy, which\nallows direct, sensitive, and reproducible monitoring of product formation in intact two-\nphase systems. These analyses are presented in Fig. 2 and Fig. 5 and summarized in the\ncorresponding Supplementary Information tables. We included the MS data is presented\nin the revised SI Fig. S6 and Fig. S13.\nReviewer #2\nThe revised manuscript by Massarano et al. has been significantly improved compared\nto the original version in terms of both novelty and experimental rigor. First, the authors\nhave reinforced the discussion part to clearly address the conceptual novelty that their\nwork has demonstrated, including intrinsic catalytic properties and Zn-dependent\nswitching between two distinct mechanisms of catalysis. (which is now also conveyed\nin the revised title). Additionally, the authors have included significant amount of\nrigorous kinetic studies, in addition to the FRAP and DLS experiments that were\noriginally missing, to demonstrate the sequence-based control over the condensate\ncatalytic and material characteristics.\nTherefore, the authors have answered all of my points and concerns provided in the first\nround of review. I believe that the manuscript is now ready for the publication, and thus\nrecommend it to be accepted. I believe that this work will provide a novel addition to the\nresearch field of LLPS, and improve our understanding of how condensate properties\ncould be precisely tuned in LLPS-based systems.\nResponse: We sincerely thank the reviewer for the positive assessment of the revised\nmanuscript. We are grateful for the recognition of the conceptual advances and overall\nsuitability of the work for publication.\nReviewer #4\nThe manuscript by Lampel and co-workers describes the formation of LLPS peptide\ncondensates capable of catalysing ester hydrolysis through either Zn²⁺-dependent or\nZn²⁺-independent mechanisms. The authors combine microscopy, kinetic\nmeasurements, and computational studies to explore how histidine residues and metal\ncoordination contribute to catalysis.\nOverall, the manuscript lacks a clear hypothesis-driven design, quantitative\nexperimental validation of the proposed mechanisms, and the level of mechanistic\ngenerality expected for the journal.\nConsidering the level of novelty of the topic and the scope of the study, I do not believe\nthat the manuscript is suitable for publication in Nature Communications, which\naddresses a broader multidisciplinary audience. A more specialised chemistry journal\nfocusing on peptide-based catalysis or supramolecular systems would likely be a more\nappropriate venue.\nResponse: See response below to comment #1.\n1. The manuscript focuses on peptide LLPS systems and their catalytic activity toward\nester hydrolysis. Although this topic remains of chemical interest, it does not represent\na particularly timely or conceptually novel advance for the Nature portfolio. Several\nstudies have already reported catalytic coacervates and peptide condensates\nemploying similar model reactions. The present work refines rather than expands the\nexisting framework and therefore does not meet the broad conceptual impact expected\nfor this journal.\nResponse: We thank the reviewer for this comment and respectfully disagree with the\nassessment that the present work represents a purely incremental refinement of existing\nstudies. While catalytic activity in coacervates and peptide condensates has been\nreported previously, the unique objective of our study is to dissect how catalytic\nperformance in peptide condensates emerges from the interplay between peptide\nsequence, metal coordination, condensate microenvironment, and internal dynamics,\nrather than from phase separation alone.\nWe acknowledge that this distinction may not have been sufficiently clear in the original\nversion of the manuscript. However, our extensive dataset, presented in both the main\ntext and the SI, provides a comprehensive characterization of each system, including\nphase behavior, material properties, Zn²⁺ accumulation, internal mobility, and fully\nquantitative kinetic analyses with appropriate controls. Importantly, the study\nestablishes two distinct catalytic regimes within the same condensate platform,\nenabling switching between a Zn²⁺-independent, histidine-driven mechanism and a Zn²⁺-\ndependent regime in which metal coordination modulates catalysis through multiple\ncoupled effects.\nTo further address this conceptual point, and in response to comments from Reviewer 1,\nwe have added substantial new analyses to the revised manuscript. Specifically, we\nquantified the pH of both the dilute and dense phases across peptide sequences and\nZn²⁺ concentrations. We found that the dense phase of all condensates is more basic\nthan the dilute phase, with condensates formed at lower ZnCl₂ concentrations (0.2 mM)\nexhibiting dense-phase pH values >9, compared to lower pH at the highest ZnCl₂\nconcentration (0.67 mM). We attribute this trend to Zn²⁺–His coordination: at low Zn²⁺,\nfewer histidine residues are coordinated, increasing the effective basicity of the dense\nphase, whereas extensive coordination at higher Zn²⁺ reduces histidine basicity and\nlowers dense-phase pH. The elevated basicity observed at low Zn²⁺ is expected to\npromote hydrolytic reactions.\nFor the Zn²⁺-free systems, both the dilute and dense phases of R2H/E and R/E were found\nto be basic, with no significant differences between them. To directly test the role of\nbasicity, we performed kinetic measurements of ester hydrolysis at pH 7.5 and 9.0 (Fig.\nS10 and S15), which confirmed that increased pH accelerates the reaction. Together with\nFRAP-derived diffusivity measurements and Zn partitioning analyses, these results\ndemonstrate that catalytic output is governed by interdependent effects of dense-phase\nbasicity, molecular mobility, and metal coordination, rather than hydrophobicity or\ncompaction alone. To highlight this important point, we created a schematic framework\nin Fig. 1c showing how the different properties of the condensates affect their catalytic\nactivity (please see response to comment #6). In addition, we reflect these insights in the\nrevised Discussion.\nWe therefore believe that the present work extends the existing framework of catalytic\nLLPS systems from descriptive observations to a mechanistic and quantitative\nunderstanding, establishing sequence-encoded and metal-regulated design principles\nfor catalytic condensates. On this basis, we therefore believe that the study provides\ninsights of broad relevance to biomolecular condensates, supramolecular catalysis, and\nemergent chemical function in soft matter systems.\n2. The rationale behind the experimental sequence requires clearer justification. The\nauthors initially state that Zn²⁺ promotes LLPS and contributes to catalysis, but the data\nindicate that higher Zn²⁺ concentrations inhibit activity. This outcome is intriguing yet\nundermines the central design logic. It remains unclear why the system was optimised\naround Zn²⁺ if Zn²⁺ coordination ultimately suppresses reactivity. The authors should\nexplicitly address whether the Zn²⁺-free system was conceived as a control or as an\nalternative catalytic regime.\nResponse: We thank the reviewer for this comment and agree that the experimental\nrationale required clarification. Our initial hypothesis, based on prior work on histidine-\ncontaining catalytic peptides, was that Zn²⁺ coordination would enhance ester hydrolysis\nwhile promoting LLPS. While Zn²⁺ indeed promotes condensate formation, we\nunexpectedly found that higher Zn²⁺ concentrations inhibit catalysis by inducing His–\nZn²⁺-mediated crosslinking, reducing internal diffusivity and limiting product (and likely\nalso substrate) partitioning.\nThis unexpected behavior motivated a focused mechanistic investigation into how metal\ncoordination can simultaneously promote phase separation while suppressing catalysis.\nImportantly, the Zn²⁺-free system was conceived not merely as a control, but as an\nalternative catalytic regime that isolates histidine-driven chemistry from metal-mediated\neffects. To clarify this point, we have revised the manuscript to explicitly frame the Zn²⁺-\nfree system as a complementary mode of catalysis.\nTo clarify this point we revised as follows:\nPage 6 line 5: “Building on previous work on esterase-mimicking catalytic peptides16, we\nused Zn2+ as a cofactor for ester hydrolysis and hypothesized that Zn2+ could function\nboth as a cofactor and an initiator of LLPS (Fig. 1).”\nPage 12 line 10: “In light of the inhibitory effect of Zn2+ on condensate catalytic capacity,\nwe next examined catalysis in the absence of ZnCl as an alternative mechanistic\n2\nregime. We first analyzed the LLPS propensity of oppositely charged peptides (at 1:1\nratio) in the absence of ZnCl …”\n2\n3. Some figure references are inconsistent or incomplete (e.g., “Fig. 1b–g” does not\ncorrespond to any figure panels). Furthermore, key datasets are represented only as\nheat maps or schematic diagrams, with no access to the raw numerical data. Providing\nthe quantitative values underlying the phase diagrams and kinetic analyses would be\nessential for reproducibility and for assessing data quality.\nResponse: As indicated in the original manuscript, all raw numerical data underlying the\nkinetic analyses were deposited in Zenodo and are accessible through the link provided\nat the end of the manuscript. To further improve transparency and reproducibility, we\nhave now included the quantitative turbidity values underlying the phase-diagram heat\nmaps in Tables S2–S4 of the revised SI. In addition, we have carefully checked and\ncorrected all figure references throughout the manuscript to ensure consistency and\naccuracy.\n4. The level of characterisation is insufficient to support the claim that bona fide LLPS\ncondensates are formed. Bright-field microscopy, DLS, and FRAP provide\nmorphological information but do not establish the molecular organisation or Zn²⁺\ncoordination environment. Complementary spectroscopic data (e.g., circular\ndichroism, FTIR, or NMR) would be required to confirm peptide structure and metal\nbinding. Without these, it remains uncertain whether the observed droplets are genuine\ndynamic condensates or partially aggregated assemblies.\nResponse: We thank the reviewer for this constructive comment. In the LLPS field, bona\nfide condensates are defined primarily by their dynamic behavior, and the most widely\naccepted criteria are the observation of droplet coalescence and the presence of internal\nmolecular mobility measured by FRAP. These functional characteristics, rather than a\nspecific spectroscopic signature, define condensates, as established in foundational\nstudies from the laboratories of Pappu, Rosen, and Brangwynne [For reference see: Dar,\nF., et al, 2024, Nature communications, 15(1), 3413; Gibson, B. A. et al, 2019, Cell, 179,\n470-484; Gibson, B.A.and Rosen, M.K., 2023. PNAS, 120, p.e2218085120; Bracha, D. et\nal, Nature biotechnology, 37(12), 1435-1445].\nTo address the reviewer’s concern directly, we performed additional CLSM experiments\nand now include time-lapse videos (Supplemental Vidoes 1-5) that clearly show fusion\nand coalescence of droplets across all peptide systems. These movies visibly\ndemonstrate the fluid nature of the assemblies and confirm that they are not partially\naggregated particles but dynamic liquid condensates. The FRAP measurements included\nin the manuscript further support this conclusion by revealing substantial recovery of\nfluorescence after photobleaching for both Zn²⁺-dependent and Zn²⁺-free condensates,\nconsistent with a mobile internal environment.\nBeyond establishing liquid behavior, we also expanded the molecular characterization of\npeptide structure and Zn²⁺ coordination by performing CD analysis. The CD analysis\nshows that the His-containing peptides retain a random-coil signature, yet exhibit\ndistinct spectral shifts upon addition of ZnCl₂. For E2H, a new maximum near 203 nm\nappears and intensifies with increasing Zn²⁺ concentration, while R2H displays a\nprogressive decrease in its random-coil minimum under the same conditions. In\ncontrast, peptides lacking His show no detectable spectral changes in the presence of\nZn²⁺, indicating that the metal interacts specifically with His-containing peptides. These\nobservations are fully consistent with Zn²⁺-His coordination contributing to LLPS and to\nthe altered material properties of the condensates.\nWe also expanded our Trp emission analysis, which reinforce these findings. His-\ncontaining peptides show strong Zn²⁺-induced increase in Trp fluorescence intensity,\nwhereas peptides without His do not. The agreement between CD and Trp emission\nresults provides an independent line of evidence that Zn²⁺ binds selectively to His-\nbearing peptides and supports the mechanistic interpretation that Zn²⁺ modulates both\ncondensate formation and the internal dynamics captured by FRAP.\nTaken together, the coalescence videos, FRAP data, CD spectra, and Trp emission\nanalyses demonstrate that the assemblies are phase separated condensates. These\nadditional datasets directly address the reviewer’s concern regarding the level of\ncharacterization and establish both the liquid nature of the droplets and the molecular\nbasis of Zn²⁺ interaction within the system.\nThe results of the CD and Trp emission analyses are presented in Fig. S12 of the revised\nSI. The following text was added to the revised MS (page 11 line 14): “To confirm that the\npeptide directly interacts with Zn2+ ions, we performed CD analysis in the absence or\npresnece of ZnCl . The CD analysis (Fig. S12) shows that the His-containing peptides R2H\n2\nand E2H retain a random-coil signature with a ~196 nm minimum and a ~225 nm\nmaximum, yet show distinct spectral shifts upon addition of ZnCl₂. For E2H, a new\nmaximum near 203 nm appears and intensifies with increasing Zn²⁺ concentration, while\nR2H displays a progressive decrease in its random-coil minimum under the same\nconditions. In contrast, peptides lacking His show no detectable spectral changes in the\npresence of Zn²⁺, indicating that the metal interacts specifically with His-containing\npeptides. In addition, we leveraged the intrinsic fluorescence of Trp within the peptides\nto perform Trp emission analysis in which environmental changes to the indole side chain\nresults in changes in the emission intensity ( =280 nm). The results show a significant\nex\nincrease in the Trp emission intensity for the His-containing peptides, R2H and E2H, in\nthe presence of 0.67 mM ZnCl while no change is observed for the peptides lacking His,\nTogether, the CD and Trp emission analysis show that Zn2+ ions interact with the His-\ncontaining peptides. These observations strengthen the phase diagrams and FRAP\nresults which show that Zn2+ promotes phase separation and slows the dense phase\ninternal mobility, likely by strong interactions with R2H and E2H.”\n5. The kinetic studies rely on a single substrate (4-MU-acetate), and the comparison to\nliterature values obtained with other substrates (e.g., p-nitrophenyl acetate) is not\nmeaningful. The mechanistic claims rely entirely on computational models, without\nexperimental validation through pH-rate profiles, isotope effects, or metal titrations.\nThese additional experiments would be essential to establish whether the observed\ncatalysis indeed proceeds through the proposed Zn²⁺-dependent and Zn²⁺-independent\npathways.\nResponse: Please see response to comment #1 and responses to Reviewer 1 comments\n#1 and #2.\n6. The manuscript would benefit from a clearer structure separating experimental\nresults from interpretation. The text is occasionally difficult to follow, and figure\ndescriptions are fragmented between the main text and the Supplementary\nInformation. The inclusion of peptide sequences or schematic representations in the\nmain figures (currently confined to Table S1) would greatly improve readability.\nI also evaluated the responses to Reviewer 3's comments. I can confirm that the\ncomments raised by Reviewer 3 have been satisfactorily addressed.\nResponse: We thank the reviewer for the comment. To address the point, we have revised\nthe text throughout the manuscript to separate the results from their interpretation\n(changes highlighted in red in the MS). Wherever suitable, we dedicated the last\nparagraph of every section in the manuscript for interpretation of the results and a short\ndiscussion of the findings.\nIn addition, following the reviewer’s comment, we have revised Figure 1 to show the\npeptide sequences. Moreover, following the other comments raised by the reviewer, we\nadded a summarizing scheme showing the relations between the different properties of\ncondensates (i.e. phase separation propensity, Zn concentration, basicity, diffusivity, and\npartitioning) and their catalytic activity. We also reflected these insights in the\nabstract."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have done a terrific job doing the revision. Congratulation for this beautiful work.", "reviewer_4": "The revised manuscript has been significantly improved compared to the original version. The authors have further strengthened the discussion to more clearly articulate the conceptual contribution of the work, including the intrinsic catalytic properties of the peptide condensates and the Zn(II)-dependent switching between two distinct catalytic regimes, which is now also reflected in the revised title. In addition, the revised manuscript includes substantial new experimental data, including expanded kinetic analyses and improved condensate characterization, together with additional controls that help clarify the physicochemical basis of the observed reactivity.\nOverall, the authors have carefully addressed my comments I believe that the manuscript is now suitable for publication."}, "author_response": ""}]

Decision letter