Liquid–liquid phase separation of proteins underpins the formation of membraneless compartments in living cells. Elucidating the molecular driving forces underlying protein phase transitions is therefore a key objective for understanding biological function and malfunction. Here we show that cellular proteins, which form condensates at low salt concentrations, including FUS, TDP-43, Brd4, Sox2, and Annexin A11, can reenter a phase-separated regime at high salt concentrations. By bringing together experiments and simulations, we demonstrate that this reentrant phase transition in the high-salt regime is driven by hydrophobic and non-ionic interactions, and is mechanistically distinct from the low-salt regime, where condensates are additionally stabilized by electrostatic forces. Our work thus sheds light on the cooperation of hydrophobic and non-ionic interactions as general driving forces in the condensation process, with important implications for aberrant function, druggability, and material properties of biomolecular condensates.

Proteins are the fundamental building blocks for high-performance materials in nature. Such materials fulfill structural roles, as in the case of silk and collagen, and can generate active structures including the cytoskeleton. Attention is increasingly turning to this versatile class of molecules for the synthesis of next-generation green functional materials for a range of applications. Protein nanofibrils are a fundamental supramolecular unit from which many macroscopic protein materials are formed. In this Review, we focus on the multiscale assembly of such protein nanofibrils formed from naturally occurring proteins into new supramolecular architectures and discuss how they can form the basis of material systems ranging from bulk gels, films, fibers, micro/nanogels, condensates, and active materials. We review current and emerging approaches to process and assemble these building blocks in a manner which is different to their natural evolutionarily selected role but allows the generation of tailored functionality, with a focus on microfluidic approaches. We finally discuss opportunities and challenges for this class of materials, including applications that can be involved in this material system which consists of fully natural, biocompatible, and biodegradable feedstocks yet has the potential to generate materials with performance and versatility rivalling that of the best synthetic polymers.

Microscale hydrogels consisting of macromolecular networks in aqueous continuous phases have received increasing attention because of their potential use in tissue engineering, cell encapsulation and for the storage and release of cargo molecules. However, for applications targeting intracellular delivery, their micrometer-scale size is unsuitable for effective cellular uptake. Nanoscale analogs of such materials are thus required for this key area. Here, we describe a microfluidics/nanofluidics-based strategy for generating monodisperse nanosized water-in-oil emulsions with controllable sizes ranging from 2500 ± 110 nm down to 51 ± 6 nm. We demonstrate that these nanoemulsions can act as templates to form protein nanogels stabilized by supramolecular fibrils from three different proteins. We further show that these nanoparticles have the ability to penetrate mammalian cell membranes and deliver intracellular cargo. Due to their biocompatibility and lack of toxicity, natural protein-based nanoparticles present advantageous characteristics as vehicles for cargo molecules in the context of pharmaceutical and biomedical applications.

Protein‐based fibers are used by nature as high‐performance materials in a wide range of applications, including providing structural support, creating thermal insulation, and generating underwater adhesives. Such fibers are commonly generated through a hierarchical self‐assembly process, where the molecular building blocks are geometrically confined and aligned along the fiber axis to provide a high level of structural robustness. Here, this approach is mimicked by using a microfluidic spinning method to enable precise control over multiscale order during the assembly process of nanoscale protein nanofibrils into micro‐ and macroscale fibers. By varying the flow rates on chip, the degree of nanofibril alignment can be tuned, leading to an orientation index comparable to that of native silk. It is found that the Young's modulus of the resulting fibers increases with an increasing level of nanoscale alignment of the building blocks, suggesting that the mechanical properties of macroscopic fibers can be controlled through varying the level of ordering of the nanoscale building blocks. Capitalizing on strategies evolved by nature, the fabrication method allows for the controlled formation of macroscopic fibers and offers the potential to be applied for the generation of further novel bioinspired materials.

Many cellular proteins have the ability to demix spontaneously from solution to form liquid condensates. These phase-separated structures form membraneless compartments in living cells and have wide-ranging roles in health and disease. Elucidating the molecular driving forces underlying liquid-liquid phase separation (LLPS) of proteins has thus become a key objective for understanding biological function and malfunction. Here we show that proteins implicated in cellular phase separation, such as FUS, TDP-43, and Annexin A11, which form condensates at low salt concentrations via homotypic multivalent interactions, also have the ability to undergo LLPS at high salt concentrations by reentering into a phase-separated regime. Through a combination of experiments and simulations, we demonstrate that phase separation in the high-salt regime is mainly driven by hydrophobic and non-ionic interactions. As such, it is mechanistically distinct from the low-salt regime, where condensates are stabilized by a broad mix of electrostatic, hydrophobic, and non-ionic forces. Our work thus expands the molecular grammar of interactions governing LLPS of cellular proteins and provides a new view on hydrophobicity and non-ionic interactions as non-specific driving forces for the condensation process, with important implications for the aberrant function, druggability, and material properties of biomolecular condensates. One Sentence SummaryProteins implicated in cellular phase separation can undergo a salt-mediated reentrant liquid-liquid phase transition.

Self-assembling peptides and proteins have the potential to serve as multifunctional building blocks for the generation of versatile materials for a wide range of biomedical applications. In particular, supramolecular hydrogels comprised of self-assembled protein nanofibrils, have been used in contexts ranging from tissue engineering to drug delivery. Due to the rapid emergence of multidrug resistant bacteria, development of biomaterials with intrinsic antimicrobial properties has been continuously increasing. Here, we describe hybrid organic/ inorganic nanofibrillar silk microgels decorated with silver nanoparticles that display potent antimicrobial activity in vitro and in vivo and are able to adhere bacterial cells to their surfaces while subsequently eradicating them, through a two-step mechanism of action. Importantly, in contrast to treatments involving conventional silver, these silk−silver microgels are nonhemolytic and noncytotoxic toward mammalian cell lines. Finally, we show that these hybrid microgels display substantial efficacy as topical antimicrobial agents in a murine model of surgical site infections.

Microfluidic devices can be used to produce single, double and higher order emulsions, where droplet sizes can be precisely controlled and modulated. Such emulsions have great potential for the storage and study of biomolecules, including peptides and proteins. However, advancement of this technique has remained challenging due to the tendency of various biomolecules to adhere to the surface of the formed channels, resulting in changes in surface wetting and fouling on the micrometer scale. Thus, precise control of surface wettability plays a crucial role in the processes that govern droplet formation. Here, we report an approach for producing both water-oil-water (w/o/w) and oil-water-oil (o/w/o) double emulsions without any need for surface modification, an enabling feature for biomolecular encapsulation. Using this strategy, we show that the number of monodisperse encapsulated internal droplets can be controlled systematically and reproducibly by suitable adjustment of the relevant flow rates, and ranges from 1 to 40 in the case of w/o/w emulsions. We further demonstrate that the number of internal droplets scales linearly with the reciprocal flow rate of the outer continuous phase, when the inner and middle phase flow rates are kept constant. We demonstrate that this approach is suitable for forming double emulsions where the inner phase consists of reconstituted silk protein solution whereby incubation of the internal droplets can be induced to form a gel resulting in silk fibroin microgels surrounded by an external oil shell. Finally, for o/w/o emulsions, we show that single or multiple monodisperse internal droplets can be encapsulated with a size that ranges over 1 order of magnitude, from ca. 10 μm to >100 μm. Moreover, o/w/o emulsions where the middle phase consists of silk fibroin solution were prepared and by allowing the protein to aggregate, a core-shell structure was formed. This microfluidic strategy allows for multiple emulsions to be generated drop by drop for biomolecular solutions with potential applications in the biomedical and pharmaceutical fields.

The aggregation of α-synuclein into amyloid fibrils has been under scrutiny in recent years because of its association with Parkinson’s disease. This process can be triggered by a lipid-dependent nucleation process, and the resulting aggregates can proliferate through secondary nucleation under acidic pH conditions. It has also been recently reported that the aggregation of α-synuclein may follow an alternative pathway, which takes place within dense liquid condensates formed through phase separation. The microscopic mechanism of this process, however, remains to be clarified. Here, we used fluorescence-based assays to enable a kinetic analysis of the microscopic steps underlying the aggregation process of α-synuclein within liquid condensates. Our analysis shows that at pH 7.4, this process starts with spontaneous primary nucleation followed by rapid aggregate-dependent proliferation. Our results thus reveal the microscopic mechanism of α-synuclein aggregation within condensates through the accurate quantification of the kinetic rate constants for the appearance and proliferation of α-synuclein aggregates at physiological pH.