Stoffel, F. | Search bioRxiv

bioRxiv2026

Engineering nanocondensate formation through sequence composition and patterning

Many biological proteins can assemble into dynamic, non-stoichiometric structures known as biomolecular condensates. Although typically observed at micrometer scales in vitro, recent evidence shows that these condensates often appear as nanoscale assemblies both in vitro and in cells. Moreover, biochemical reactions can be more efficiently promoted in nanoscale condensates than in micron-sized droplets, due to mass-transfer limitations and interfacial effects. Therefore, in analogy with colloids, the function of condensate materials can be engineered by tuning their size distribution. However, controlling the size of condensates remains challenging, as the molecular mechanisms that prevent small condensates from coarsening into larger ones are still poorly understood. Here, we developed and applied a computational pipeline that combines high-throughput molecular simulations, machine learning, and mixed-integer linear programming to design phase-separating peptides that form metastable nanocondensates across a broad range of experimental conditions. In addition to experimentally validating these peptides, we elucidate the underlying molecular mechanisms and derive initial design rules. In particular, we show that scaffold net charge combined with sequence blockiness can lead to high phase separation propensity while simultaneously yielding low interfacial tension, thereby slowing ripening. Moreover, these combined properties induce an electrostatics-driven alignment of molecules at the interface, which generates an additional size-dependent coalescence barrier. We further show that these features are shared by biological proteins, providing a possible mechanistic basis for the widespread occurrence of nanocondensates in biological systems. Altogether, our findings shed light on the molecular mechanisms behind nanocondensate formation, and provide a platform to design nanocondensates for several potential applications in bioengineering and biotechnology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/706365v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@17c379aorg.highwire.dtl.DTLVardef@26686dorg.highwire.dtl.DTLVardef@19206a7org.highwire.dtl.DTLVardef@1f2da71_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Enhancement of Enzymatic Activity by Biomolecular Condensates through pH Buffering

Biomolecular condensates can affect enzymatic reactions by locally changing not only concentrations of molecules but also their environment. Since protein conformations can differ between the dense and dilute phase, phase separation can particularly modulate enzymes characterized by a conformation-dependent activity. Here, we generate enzymatic condensates based on a lipase from Bacillus thermocatenulatus, which exhibits an equilibrium between a closed, inactive state, and an open, active conformation. We show that the activity of the enzyme increases inside the dense phase, leading to an enhancement of the overall reaction rate in the phase-separated system. Moreover, we demonstrate that these condensates can generate a more basic environment compared to the surrounding solution. As a consequence, the phase-separated system maintains a high enzymatic activity even in acidic conditions that would be otherwise less favourable for the lipase. We further show that the formation of two phases with distinct pH values optimizes a cascade reaction involving two enzymes with different optimal pH conditions. Our results demonstrate that biomolecular condensates can also affect the dependence of enzymatic rates on solution pH, thereby expanding the accessible pH interval and enabling network reactions with enzymes that require distinct pH values. These findings have crucial implications in biology and biotechnology for biocatalytic engineering.

biochemistry↗