Coacervation-enhanced peptide catalysis

Now, Ana S. Pina and colleagues report that LLPS can significantly increase the catalytic activity of peptides. Specifically, the researchers created single peptide-based coacervates, whereby the spatial confinement led to a more folded structure of the peptides (pictured). The peptide P7 (KVYFSIPWRVPM-NH₂), which was used as a model system, has a high affinity for phosphorylated assemblies, can hydrolyse phosphate ester molecules, and contains arginine, lysine, serine and proline residues that are recognized to be beneficial for phase separation. Screening of peptide and salt concentrations and different temperatures led to suitable conditions to induce the formation of P7 coacervates and further optimization of the experimental design reduced unwanted reactions such as aggregation and precipitation of the peptides and division of the coacervates. Spectroscopic methods were then used to investigate the effect of LLPS on peptide conformation. It was shown that the compartmentalization via LLPS led to a stabilization of the secondary structure of P7. Specifically, a fully folded β-hairpin structure was detected compared to a flexible hairpin-like peptide in solution. Moreover, charge and hydrophobicity of the peptide influenced the partitioning of guest molecules by the coacervates. It was further shown that phosphorylation had an even more pronounced effect, leading to preferential uptake compared to non-phosphorylated versions of protein molecules. Subsequently, the coacervates were tested for catalytic phosphate ester hydrolysis of p-nitrophenyl phosphate (pNPP) as the substrate and catalytic parameters of k_cat = (4.9 ± 0.6) × 10^–3 s⁻¹ and K_M = (8.2 ± 3.2) × 10^–4 M and a catalytic efficiency of k_cat/K_M = 5.9 ± 0.2 were determined. Impressively, this constitutes a 15,000-fold improvement in catalytic efficiency over the reaction mediated by the free peptides in solution determined in a prior work.

The demonstration that simple, small peptides can enable self-coacervation, selectively recruit substrates and accelerate catalysis will likely be of interest to origin of life researchers and might also serve as an inspiration for the future design of systems for drug delivery and sensing.