Understanding the relationship between an amino acid sequence and its phase separation has important implications for analyzing cellular function, treating disease, and designing novel biomaterials. Several sequence features have been identified as drivers for protein liquid-liquid phase separation (LLPS), leading to the development of a “molecular grammar” for LLPS. In this work, we further probed how sequence modulates phase separation and the material properties of the resulting condensates. Specifically, we used a model intrinsically disordered polypeptide composed of an 8-residue repeat unit and performed systematic sequence manipulations targeting sequence features previously overlooked in the literature. We generated sequences with no charged residues, high net charge, no glycine residues, or devoid of aromatic or arginine residues. We report that all but one of the twelve variants we designed undergo LLPS, albeit to different extents, despite significant differences in composition. These results support the hypothesis that multiple interactions between diverse residue pairs work in tandem to drive phase separation. Molecular simulations paint a picture of underlying molecular details involving various atomic interactions mediated by not just a handful of residue types, but by most residues. We characterized the changes to inter-residue contacts in all the sequence variants, thereby developing a more complete understanding of the contributions of sequence features such as net charge, hydrophobicity, and aromaticity to phase separation. Further, we find that all condensates formed behave like viscous fluids, despite large differences in their viscosities. The results presented in this study significantly advance the current sequence-phase behavior and sequence-material properties relationships to help interpret, model, and design protein assembly.

Heterogeneities in hydrogel scaffolds are known to impact the performance of cells in cell-laden materials constructs, and we have employed the phase separation of resilin-like polypeptides (RLPs) as a means to generate such materials.Here, we study the compositional features of resilin-like polypeptides (RLPs) that further enable our control of their liquid−liquid phase separation (LLPS) and how such control impacts the formation of microstructured hydrogels. The evaluation of the phase separation of RLPs in solutions of ammonium sulfate offers insights into the sequence-dependent LLPS of the RLP solutions, and atomistic simulations, along with 2D-nuclear Overhauser effect spectroscopy (NOESY) and correlated spectroscopy (COSY) 1 H NMR, suggest specific amino acid interactions that may mediate this phase behavior. The acrylamide functionalization of RLPs enables their photo-cross-linking into hydrogels and also enhances the phase separation of the polypeptides. A heating−cooling protocol promotes the formation of stable emulsions that yield different microstructured morphologies with tunable rheological properties. These findings offer approaches for choosing RLP compositions with phase behaviors that can be easily tuned with differences in temperature to control the resulting morphology and mechanical behavior of the heterogeneous hydrogels in regimes useful for biological applications.

Hydrogels have been broadly studied for applications in clinically motivated fields such as tissue regeneration, drug delivery, and wound healing, as well as in a wide variety of consumer and industry uses. While the control of mechanical properties and network structures are important in all of these applications, for regenerative medicine applications in particular, matching the chemical, topographical and mechanical properties for the target use/tissue is critical. There have been multiple alternatives developed for fabricating materials with microstructures with goals of controlling the spatial location, phenotypic evolution, and signaling of cells. The commonly employed polymers such as poly(ethylene glycol) (PEG), polypeptides, and polysaccharides (as well as others) can be processed by various methods in order to control material heterogeneity and microscale structures. We review here the more commonly used polymers, chemistries, and methods for generating microstructures in biomaterials, highlighting the range of possible morphologies that can be produced, and the limitations of each method. With a focus in liquidliquid phase separation, methods and chemistries well suited for stabilizing the interface and arresting the phase separation are covered. As the microstructures can affect cell behavior, examples of such effects are reviewed as well.

Local, micromechanical environment is known to influence cellular function in heterogeneous hydrogels, and knowledge gained in this area will facilitate the improved design of biomaterials for tissue regeneration. In this study, we utilize a system comprising microstructured resilin-like polypeptide (RLP)-poly(ethylene glycol) (PEG) hydrogels and hydrogels predominantly comprising either RLP-rich or PEG-rich phases as controls. The micromechanical properties of RLP-PEG microstructured hydrogels were evaluated with oscillatory shear rheometry, compression DMA, smallstrain microindentation, and large-strain indentation and puncture over a range of different deformation length scales. The measured elastic moduli were consistent with volume averaging models, indicating that volume fraction, not domain size, plays a dominant role in determining the low strain mechanical response. Large-strain indentation under a confocal microscope enabled the visualization of the microstructured hydrogel micromechanical deformation, emphasizing the translation, rotation, and deformation of RLP-rich domains. The fracture initiation energy values obtained from puncture experiments demonstrated that failure of the composite hydrogels was controlled by the RLP-rich phase, and their independence with domain size suggested that failure initiation was controlled by multiple domains within the strained volume. Our approach and findings provide new quantitative insight into the micromechanical response of soft hydrogel composites on length scales relevant to those of biological cells and highlight the opportunities in employing these methods to understand the physical origins of mechanical properties of soft synthetic and biological materials.