Dynamic, protein-rich molecular assemblies are ubiquitous and versatile biological structures. However, our understanding of these organized structures, as well as our ability to synthetically replicate them, are lacking due to the difficulty in recreating their complex physicochemical makeup. Here we present a simple method for programming diverse molecular assemblies comprised of intrinsically disordered proteins possessing sequences of low-complexity (SLC) within microenvironments. By encoding the stimulus-induced phase behavior of protein mixtures, we demonstrate the reversible formation of a variety of protein-rich architectures, ranging from uniform nano-, meso-, and micro-scale puncta (small, distinct particulates) to orthogonal, multilayered granules.
Thermally responsive elastin-like polypeptides (ELPs) are prototypical building blocks for the reconstitution of cell-free protein granule assays because of their compositional and structural similarity to the native proteins involved in cellular assemblies. These intrinsically disordered proteins are comprised of pentameric repeats possessing SLC: Val-Pro-Gly-Xaa-Gly, where Xaa is a guest residue. We developed an in vitro platform to resolve the protein sequence-property relationship within the context of liquid-liquid phase behavior and on resultant granule formation by creating microfluidic generated water droplets with a length scale similar to that of a cell containing multiple ELP components. We then controllably triggered ELP phase transitions within the droplets to generate a number of granule-like structures with controllable architecture, size and composition.
We show the ability to reversibly self-assemble uniform granules of controllable number, size, and hierarchical multilayered structure by programming protein amino acid sequence and phase transition phenomena. We anticipate that the ability to build protein assemblies via programmable phase separation will facilitate new insights on (1) the genetic to molecular to macroscale relationship of phase separating proteins and their assembly into granules, (2) engineered protein-based hydrogels, and (3) the physics of protein phase separation.