Sequence Context and Complex Hofmeister Salt Interactions Dictate Phase Separation Propensity of Resilin-like Polypeptides.

Resilin is an elastic material found in insects with exceptional durability, resilience, and extensibility, making it a promising biomaterial for tissue engineering. The monomeric precursor, pro-resilin, undergoes thermo-responsive self-assembly through liquid-liquid phase separation (LLPS). Understanding the molecular details of this assembly process is critical to developing complex biomaterials. The present study investigates the interplay between the solvent, sequence syntax, structure, and dynamics in promoting LLPS of resilin-like-polypeptides (RLPs) derived from domains 1 and 3 of Drosophila melanogaster pro-resilin. NMR, UV-vis, and microscopy data demonstrate that while kosmotropic salts and low pH promote LLPS, the effects of chaotropic salts with increasing pH are more complex. Subtle variations between the repeating amino acid motifs of resilin domain 1 and domain 3 lead to significantly different salt and pH dependence of LLPS, with domain 3 sequence motifs more strongly favoring phase separation under most conditions. These findings provide new insight into the molecular drivers of RLP phase separation and the complex roles of both RLP sequence and solution composition in fine-tuning assembly conditions.