Peptide-Based Coacervates as Catalytic Microreactors

Enzymes' machinery has been hypothesized to be evolved from much simpler functional precursors such as peptides (<i>1</i>). Peptides are well-implemented as chiral catalysts in organic reactions (<i>2</i>). However, their use in aqueous reactions is still limited and remains a challenge in molecular engineering. However, the modest efficiency of peptide catalysts in aqueous media could arise from their heterogeneity in conformation, which is challenging to control (<i>3</i>). Different strategies have been used to tackle peptides' conformational heterogeneity, yet their catalytic efficiency is still modest(<i>4</i>).<br/>Cells organize their biochemical reactions in membraneless compartments, formed by liquid-liquid phase separation (LLPS) of intrinsically disordered proteins by specific peptide motifs or by their interaction with nucleic acids. Coacervates, also created by LLPS, are associated with the origin of life as primitive models of protocells (<i>5</i>) and provide mechanisms for shielding oligomers from bulk solutions and concentrating biomolecules, thus facilitating a variety of processes such as catalytic function(<i>5</i>). Therefore, the compartmentalization of catalytic peptides seems an exciting route to evolve catalytic role in often modest peptides and a strategy to constrain their conformational flexibility often observed in bulky solutions(<i>4</i>).<br/>In this work, we show for the first time the creation of dynamic micro-sized liquid condensates formed by a catalytic peptide, whose primary sequence (KVYFSIPWRVPM) (<i>6</i>) is composed of phase-separating residues (Arg, Lys, Tyr, Phe, Try, and Pro). Simultaneously, the peptide can hydrolyze phosphate ester compounds and bind to phosphotyrosine assemblies with supramolecular selectivity(<i>6</i>).<br/><br/>Sequence-structure-function relationship is critical for the formation of these catalytic coacervates. Turbidity measurements and optical microscopy revealed that the peptide could phase separate and form coacervates. However, a delicate balance between peptide concentration and environmental conditions could also lead to peptide aggregates. Circular Dichroism and NMR revealed that the peptide presents a fully-folded β-hairpin structure only in the coacervate phase, and the LLPS driven by cation-π and aromatic interactions. Compared with peptide soluble and aggregated states, the peptide shows a partially folded hairpin-like structure or a lack of structure. The dynamics and sequestration ability of the coacervates towards a range of guest molecules ranging from small molecules to phosphorylated assemblies was exploited. The partitioning of the molecules showed to be controlled by charge interactions. However, the hydrophobicity character of the catalytic peptides seems to play a role in mediating this process, which further influences the microenvironment of the catalytic coacervates.<br/><br/>Ultimately, these catalytic coacervates-based reactors' efficiency was studied and compared to the peptide in bulky solution. This work provides a substantial opportunity to leverage the field of catalytic peptides to regulate catalytic efficiency through the compartmentalization of short peptide catalysts in aqueous media.<br/><br/>1. M. Frenkel-Pinter, M. Samanta, G. Ashkenasy, L. J. Leman, <i>Chemical Reviews</i> <b>120</b>, 4707-4765 (2020).<br/>2. A. J. Metrano, S. J. Miller <i>Accounts of Chemical Research</i> <b>52</b>, 199-215 (2019).<br/>3. A. J. Metrano<i> et al,</i>. <i>Journal of the American Chemical Society</i> <b>139</b>, 492-516 (2017).<br/>4. S. Carvalho, D. Q. P. Reis, S. V. Pereira, D. Kalafatović, A. S. Pina, <i>Israel Journal of Chemistry</i>, <b> 62,</b> e202200029 (2022).<br/>5. S. Mann, Life as a Nanoscale Phenomenon. <i>Angewandte Chemie International Edition</i> <b>47</b>, 5306-5320 (2008).<br/>6. A. S. Pina<i> et al.</i>, Discovery of phosphotyrosine-binding oligopeptides with supramolecular target selectivity. <i>Chemical Science</i> <b>13</b>, 210-217 (2022).