Fisher, R. S. | Search bioRxiv

bioRxiv2025

Formation and Gelation of Elastin-like Polypeptide Complex Coacervates

Protein liquid-liquid phase separation underlies the formation of membraneless organelles in cells and performs a key role in the assembly process of natural materials such as the assembly of tropoelastin into elastic fibers. Here, we engineered a series of charged elastin-like polypeptides (ELPs) that form complex coacervates, providing a rapid method to concentrate proteins into a fluid state. Compared to coacervates formed from simple coacervation, complex coacervates exhibited greater fluidity, likely due to differences between electrostatic interactions and hydrophobic forces. We designed these ELPs to further contain crosslinking domains compatible with tyrosinase or transglutaminase and found that crosslinking was enhanced when proteins were in a complex coacervate compared to free in solution. Crosslinking the ELP complex coacervates led to the formation of gels with distinct properties dependent on the nature of the crosslinking. This work expands the design space of ELP hydrogels, offering a novel strategy for forming crosslinked networks from complex coacervates and providing opportunity for future use in tissue engineering and biocompatible biomaterials applications.

synthetic biology↗

bioRxiv2023

Tuning materials properties of globular protein-based condensates

The phase separation of biomolecules into biomolecular condensates has emerged as a ubiquitous cellular process. Understanding how intrinsically disordered protein sequence controls condensate formation and material properties has provided fundamental biological insights and led to the development of functional synthetic condensates. While these studies provide a valuable framework to understand subcellular organization via phase separation they have largely ignored the presence of folded domains and their impact on condensate properties. We set out to determine how the distribution of sticker interactions across a globular protein contributes to rheological properties of condensates and to what extent globular protein-containing condensates differ from those formed from two disordered components. We designed three variants of green fluorescent protein with different charge patterning and used dynamic light scattering microrheology to measure the viscoelastic spectrum of coacervates formed with poly-lysine over a timescale of 10-6 to 10 seconds, elucidating the response of protein condensates in this range for the first time. We further showed that the phase behavior and rheological characteristics of the condensates varied as a function of both protein charge distribution and polymer/protein ratio, behavior that was distinct to condensates formed with folded domains. Together, this work enhances our fundamental understanding of dynamic condensed biomaterials across biologically relevant length- and time-scales.

biophysics↗