Dynamic protein-rich intracellular structures that contain phase-separated intrinsically disordered proteins (IDPs) composed of sequences of low complexity (SLC) have been shown to serve a variety of important cellular functions, which include signalling, compartmentalization and stabilization. However, our understanding of these structures and our ability to synthesize models of them have been limited. We present design rules for IDPs possessing SLCs that phase separate into diverse assemblies within droplet microenvironments. Using theoretical analyses, we interpret the phase behaviour of archetypal IDP sequences and demonstrate the rational design of a vast library of multicomponent protein-rich structures that ranges from uniform nano-, meso- and microscale puncta (distinct protein droplets) to multilayered orthogonally phase-separated granular structures. The ability to predict and program IDP-rich assemblies in this fashion offers new insights into (1) genetic-to-molecular-to-macroscale relationships that encode hierarchical IDP assemblies, (2) design rules of such assemblies in cell biology and (3) molecular-level engineering of self-assembled recombinant IDP-rich materials.
Strong, tough, stretchable, and self-adhesive hydrogels are designed with intrinsically unstructured proteins. The extraordinary mechanical properties exhibited by these materials are enabled by an integration of toughening mechanisms that maintain high elasticity and dissipate mechanical energy within the protein networks.
Post-translational modification of proteins is a strategy widely used in biological systems. It expands the diversity of the proteome and allows for tailoring of both the function and localization of proteins within cells as well as the material properties of structural proteins and matrices. Despite their ubiquity in biology, with a few exceptions, the potential of post-translational modifications in biomaterials synthesis has remained largely untapped. As a proof of concept to demonstrate the feasibility of creating a genetically encoded biohybrid material through post-translational modification, we report here the generation of a family of three stimulus-responsive hybrid materials-fatty-acid-modified elastin-like polypeptides-using a one-pot recombinant expression and post-translational lipidation methodology. These hybrid biomaterials contain an amphiphilic domain, composed of a β-sheet-forming peptide that is post-translationally functionalized with a C alkyl chain, fused to a thermally responsive elastin-like polypeptide. They exhibit temperature-triggered hierarchical self-assembly across multiple length scales with varied structure and material properties that can be controlled at the sequence level.
Highlights d New engineered phase-separating IDPs capable of binding RNA d IDPs co-phase-separate with RNAs into liquid granules in response to heat d IDP-RNA-rich granules regulate translation through mRNA sequestration d Droplet-based protocells temporally inhibit translation in a programmable manner
Noncovalently cross-linked networks are attractive hydrogel platforms because of their facile fabrication, dynamic behavior, and biocompatibility. The majority of noncovalently cross-linked hydrogels, however, exhibits poor mechanical properties, which signifi cantly limit their utility in load bearing applications. To address this limitation, hydrogels are presented composed of micelles created from genetically engineered, amphiphilic, elastin-like polypeptides that contain a relatively large hydrophobic block and a hydrophilic terminus that can be cross-linked through metal ion coordination. To create the hydrogels, heat is fi rstly used to trigger the self-assembly of the polypeptides into monodisperse micelles that display transition metal coordination motifs on their coronae, and subsequently cross-link the micelles by adding zinc ions. These hydrogels exhibit hierarchical structure, are stable over a large temperature range, and exhibit tunable stiffness, self-healing, and fatigue resistance. Gels with polypeptide concentration of 10%, w/v, and higher show storage moduli of ≈1 MPa from frequency sweep tests and exhibit self-healing within minutes. These reversibly cross-linked, hierarchical hydrogels with enhanced mechanical properties have potential utility in a variety of biomedical applications.
Hydrogel particles are versatile materials that provide exquisite, tunable control over the sequestration and delivery of materials in pharmaceutics, tissue engineering, and photonics. The favorable properties of hydrogel particles depend largely on their size, and particles ranging from nanometers to micrometers are used in different applications. Previous studies have only successfully fabricated these particles in one specific size regime and required a variety of materials and fabrication methods. A simple yet powerful system is developed to easily tune the size of polypeptide-based, thermoresponsive hydrogel particles, from the nano- to microscale, using a single starting material. Particle size is controlled by the self-assembly and unique phase transition behavior of elastin-like polypeptides in bulk and within microfluidic-generated droplets. These particles are then stabilized through ultraviolet irradiation of a photo-crosslinkable unnatural amino acid (UAA) cotranslationally incorporated into the parent polypeptide. The thermoresponsive property of these particles provides an active mechanism for actuation and a dynamic responsive to the environment. This work represents a fundamental advance in the generation of crosslinked biomaterials, especially in the form of soft matter colloids, and is one of the first demonstrations of successful use of UAAs in generating a novel material.
The controllable production of microparticles with complex geometries is useful for a variety of applications in materials science and bioengineering. The formation of intricate microarchitectures typically requires sophisticated fabrication techniques such as flow lithography or multiple-emulsion microfluidics. By harnessing the molecular interactions of a set of artificial intrinsically disordered proteins (IDPs), we have created complex microparticle geometries, including porous particles, core-shell and hollow shell structures, and a unique 'fruits-on-a-vine' arrangement, by exploiting the metastable region of the phase diagram of thermally responsive IDPs within microdroplets. Through multi-site unnatural amino acid (UAA) incorporation, these protein microparticles can also be photo-crosslinked and stably extracted to an all-aqueous environment. This work expands the functional utility of artificial IDPs as well as the available microarchitectures of this class of biocompatible IDPs, with potential applications in drug delivery and tissue engineering.
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