Surface Passivation Method for the Super-repellence of Aqueous Macromolecular Condensates.

Solutions of macromolecules can undergo liquid-liquid phase separation to form droplets with ultralow surface tension. Droplets with such low surface tension wet and spread over common surfaces such as test tubes and microscope slides, complicating in vitro experiments. The development of a universal super-repellent surface for macromolecular droplets has remained elusive because their ultralow surface tension requires low surface energies. Furthermore, the nonwetting of droplets containing proteins poses additional challenges because the surface must remain inert to a wide range of chemistries presented by the various amino acid side chains at the droplet surface. Here, we present a method to coat microscope slides with a thin transparent hydrogel that exhibits complete dewetting (contact angles θ ≈ 180°) and minimal pinning of phase-separated droplets in aqueous solution. The hydrogel is based on a swollen matrix of chemically cross-linked polyethylene glycol diacrylate of molecular weight 12 kDa (PEGDA), and can be prepared with basic chemistry laboratory equipment. The PEGDA hydrogel is a powerful tool for in vitro studies of weak interactions, dynamics, and the internal organization of phase-separated droplets in aqueous solutions.

The interface of condensates of the hnRNPA1 low-complexity domain promotes formation of amyloid fibrils.

The maturation of liquid-like protein condensates into amyloid fibrils has been associated with several neurodegenerative diseases. However, the molecular mechanisms underlying this liquid-to-solid transition have remained largely unclear. Here we analyse the amyloid formation mediated by condensation of the low-complexity domain of hnRNPA1, a protein involved in amyotrophic lateral sclerosis. We show that phase separation and fibrillization are connected but distinct processes that are modulated by different regions of the protein sequence. By monitoring the spatial and temporal evolution of amyloid formation we demonstrate that the formation of fibrils does not occur homogeneously inside the droplets but is promoted at the interface of the condensates. We further show that coating the interface of the droplets with surfactant molecules inhibits fibril formation. Our results reveal that the interface of biomolecular condensates of hnRNPA1 promotes fibril formation, therefore suggesting interfaces as a potential novel therapeutic target against the formation of aberrant amyloids mediated by condensation.

Programmable Zwitterionic Droplets as Biomolecular Sorters and Model of Membraneless Organelles.

Increasing evidence indicates that cells can regulate biochemical functions in time and space by generating membraneless compartments with well-defined mesoscopic properties. One important mechanism underlying this control is simple coacervation driven by associative disordered proteins that encode multivalent interactions. Inspired by these observations, programmable droplets based on simple coacervation of responsive synthetic polymers that mimic the "stickers-and-spacers" architecture of biological disordered proteins are developed. Zwitterionic polymers that undergo an enthalpy-driven liquid-liquid phase separation process and form liquid droplets that remarkably exclude most molecules are developed. Starting from this reference material, different functional groups in the zwitterionic polymer are progressively added to encode an increasing number of different intermolecular interactions. This strategy allowed the multiple emerging properties of the droplets to be controlled independently, such as stimulus-responsiveness, polarity, selective uptake of client molecules, fusion times, and miscibility. By exploiting this high programmability, a model of cellular compartmentalization is reproduced and droplets capable of confining different molecules in space without physical barriers are generated. Moreover, these biomolecular sorters are demonstrated to be able to localize, separate, and enable the detection of target molecules even within complex mixtures, opening attractive applications in bioseparation, and diagnostics.

Sequestration within biomolecular condensates inhibits Aß-42 amyloid formation.

Biomolecular condensates are emerging as an efficient strategy developed by cells to control biochemical reactions in space and time by locally modifying composition and environment. Yet, local increase in protein concentration within these compartments could promote aberrant aggregation events, including the nucleation and growth of amyloid fibrils. Understanding protein stability within the crowded and heterogeneous environment of biological condensates is therefore crucial, not only when the aggregation-prone protein is the scaffold element of the condensates but also when proteins are recruited as client molecules within the compartments. Here, we investigate the partitioning and aggregation kinetics of the amyloidogenic peptide Abeta42 (Aß-42), the peptide strongly associated with Alzheimer's disease, recruited into condensates based on low complexity domains (LCDs) derived from the DEAD-box proteins Laf1, Dbp1 and Ddx4, which are associated with biological membraneless organelles. We show that interactions between Aß-42 and the scaffold proteins promote sequestration and local increase of the peptide concentration within the condensates. Yet, heterotypic interactions within the condensates inhibit the formation of amyloid fibrils. These results demonstrate that biomolecular condensates could sequester aggregation-prone proteins and prevent aberrant aggregation events, despite the local increase in their concentration. Biomolecular condensates could therefore work not only as hot-spots of protein aggregation but also as protective reservoirs, since the heterogenous composition of the condensates could prevent the formation of ordered fibrillar aggregates.

Adaptive Chemoenzymatic Microreactors Composed of Inorganic Nanoparticles and Bioinspired Intrinsically Disordered Proteins.

The assembly of protein and inorganic nanoparticles represents an attractive approach to generate composite materials with multiple functions. Herein, we functionalize inorganic nanoparticles with intrinsically disordered protein domains associated with the formation of membraneless compartments in cells. These protein sequences, defined as low complexity domains (LCDs), encode intermolecular interactions that drive highly controlled, dynamic self-assembly in response to environmental changes. We show that the properties of the LCDs can be transferred to inorganic nanoparticles, inducing controlled phase separation that is dynamic and responsive to ionic strength and pH. Specifically, we hybridize magnetic nanoparticles with multi-domain proteins consisting of LCD domains and a globular enzyme, generating dynamic protein-composite compartments that locally confine hybrid chemoenzymatic reactions and respond to external magnetic fields and changes in solution conditions.

Multifunctional Protein Materials and Microreactors using Low Complexity Domains as Molecular Adhesives.

Recent findings indicate that a class of disordered amino acid sequences promotes functional phase transition of biomolecules in nature. Such sequences consist of low complexity domains (LCDs) that are rich in specific amino acids. In this work, we exploit these sequences by conjugating them to soluble globular domains to develop molecular adhesives that enable sensitive, controlled self-assembly of these proteins into supramolecular architectures. In particular, we used the enzyme adenylate kinase and the green fluorescent protein as soluble domains, and we show that the addition of low complexity regions induces the formation of protein particles via a multistep process. This multistep pathway involves an initial liquid-liquid phase transition, which creates protein-rich droplets that mature into protein aggregates over time. These protein aggregates consist of permeable structures that maintain activity and release active soluble proteins. We show that the LCDs dictate specific noncovalent intermolecular interactions and phase properties that are largely independent of the given globular domain. We further demonstrate that this feature, together with the dynamic state of the initial dense liquid phase, allows one to directly assemble different globular domains within the same architecture, thereby enabling the generation of both static multifunctional biomaterials and dynamic microscale bioreactors.