Biomolecular condensates are unique structures that form in living cells through liquid-liquid phase separation (LLPS), driven primarily by intrinsically disordered proteins (IDPs) [1]. These proteins regulate biological functions by reversibly compartmentalizing molecules in response to stimuli. Synthetic condensate engineering aims to develop novel biomolecular condensates by understanding how amino acid sequences influence the mechanical properties and assembly of these IDPs. To achieve this, intrinsically disordered protein polymers (IDPPs) with a lower critical solution temperature (LCST) are often employed [2].

In this study, we focus on the formation of synthetic condensates using elastin-like recombinamers (ELRs) and examine the forces driving their coacervation, which include both simple coacervation (hydrophobic interactions) and complex coacervation (electrostatic interactions) at inter- and intramolecular levels. An IDPPs library, based on repeats of motifs found in the intrinsically disordered regions of tropoelastin, was recombinantly produced [3]. It includes two monoblocks with highly charged domains (one enriched in Glu, negatively charged, and the other in Lys, positively charged), a diblock combining these two monoblocks, and two hydrophobic control constructs containing VPGVG domains of different lengths. The latter three ELRs retained the elastin-like phase behavior, undergoing a reversible inverse temperature transition (ITT) above a characteristic transition temperature (Tt) and exhibiting random coiled coils and β-turn conformations similar to tropoelastin[4]. By contrast, the two highly charged monoblocks did not coacervate on their own.

However, mixing these charged monoblocks resulted in complex coacervation. As the molar ratio approached charge neutrality, the inverse transition temperatures decreased, and the random coil conformation shifted to include more β-turn structures, as confirmed by differential scanning calorimetry, ITC, circular dichroism, and molecular dynamics. The combined electrostatic and hydrophobic interactions produced higher-order conformations. Furthermore, the diblock system formed a more ordered structure with a lower Tt than the simple monoblock mixture.
We visualized these coacervation processes through two approaches: (1) in bulk solution and (2) in protocell systems confined within microfluidic devices. Optical and confocal microscopy showed how tuning IDPP chains affects the formation of different coacervates and revealed the critical role of inter- and intramolecular interactions in achieving successful coacervation. These imaging techniques also provided detailed insights into the dynamic behavior of the forming structures, underscoring the intricate balance of interactions required for coacervation.
Overall, our results highlight the interplay of self-organizing forces in creating complex hierarchical assemblies from IDPs. By fine-tuning inter- and intramolecular electrostatic interactions, we can govern the formation and maturation of protein condensates. These findings deepen our understanding of the self-organizing nature of biological macromolecules and LLPS, ultimately facilitating the design of complex organelle-like structures for diverse applications with significant implications in synthetic biology
[1,5,6].