Silk coatings have significant application potential in medical materials due to their biocompatibility. Through processing of silk materials and functionalization, new coating properties can be gained. Following initial protein adsorption on a surface, an ethanol post-treatment can be used to enhance properties. Ethanol is thought to induce changes in protein structure, but very little is known on its effect on the binding stability and retention of coatings. Here, we study the effect of ethanol post-treatment and substrate material on recombinant silk coating adhesion, stability, structural organization, and wettability. For critical information on binding, we use tritium labeling to accurately quantify the bound silk layer. Further, scanning electron microscopy, optical microscopy, spectroscopy techniques, and static contact angle measurements are used for detailed analysis of surface-bound protein. Ethanol is shown to affect properties widely throughout the entire silk coating. It significantly increases binding stability on all tested substrates and improves adhesion to the substrate. Ethanol also renders otherwise hydrophilic coatings more hydrophobic. Differences in the morphology of silk sublayer organization due to substrate material are also observed. Interestingly, no significant change in secondary structure is observed to explain the changes in properties observed. The presence of salt complicates the coating arrangement due to the appearance of salt crystals but enables even higher retention and larger changes in wettability. Our work complements existing knowledge of ethanol post-treatment of silk coatings with important quantification of binding, which offers new insight and provides steps for improved durability of coatings in medical applications.

Abstract Fungal surfaces must remain hydrophobic to enable growth, dispersal, and survival under fluctuating environmental conditions, yet the molecular basis of this property remains incompletely understood. Here, we identify fungisporins, fusahexins, and related cyclic non-ribosomal peptides (NRPs) as members of a conserved functional class of fungal metabolites, termed WAter Repellent Peptides (WARPs), that are required for fungal surface hydrophobicity. Across filamentous fungi, WARPs vary substantially in sequence and length but share conserved structural features, including cyclization, hydrophobic amino acid composition, and alternating D- and L-configurations, consistent with a flexible amphiphilic scaffold. Loss of WARP-producing non-ribosomal peptide synthetases results in rapid collapse of aerial hyphae upon water exposure, demonstrating that these peptides are required for maintenance of hydrophobic aerial structures. Using phage-display-derived antibodies, we localize WARPs to the hyphal surface, supporting their role as surface-associated structural components. Together, these findings identify a conserved NRPS-encoded peptide system that contributes to fungal hydrophobicity and establish WARPs as a broadly distributed class of surface-associated metabolites with structural function in filamentous fungi.

The growing use of timber in construction has created an urgent need for high-performing engineered wood. Laminating timber facilitates production of structural components, but strong interfacial bonding is essential for engineered wood to outperform solid wood. Here we introduce a method for achieving strong wood bonding using an ionic liquid-dissolved cellulose solution. At the bonding interface, the dissolved cellulose fills the lumina and entangles with the wood cell wall, forming a dense cellulose network interconnecting with wood upon regeneration in water. Concurrent hot-pressing forms a permanently interlocked structure of wood cells. The multiscale bonded interface is water resistant with a shear strength over 20 MPa, nearly twice that of solid wood. This work presents an eco-friendly, high-performing wood bonding mechanism with promising applications in engineered wood products.

Accurately quantifying specific proteins from complex mixtures like cell lysates, for example, during in vivo studies, is difficult, especially for aggregation-prone proteins. Herein, we describe the development of a specific protein quantification method that combines a solid-state dot blot approach with radiolabel detection via liquid scintillation counting. The specific detection with high sensitivity is achieved by using the Twin-Strep protein affinity tag and tritium-labeled 3HStrep-TactinXT probe. While the assay was developed with the recombinant silk protein CBM-AQ12-CBM as a target, the method can be adapted to other recombinant proteins. Variations of the protein tag and Strep-Tactin probe were tested, and it was found that only the combination of Strep-TactinXT and Twin-Strep-tag performed adequately: with this combination, a precision of 95% and an accuracy of 86% were achieved with a linear region from 19 to 400 ng and a limit of quantification at 0.4 pmol. To achieve this, critical optimization steps were preventing nonspecific adsorption and promoting surface adhesion of the target protein to the solid nitrocellulose membrane. The often-overlooked challenges of sample preparation and protein immobilization in quantification assays are discussed and insights into overcoming such issues are provided.

Many biological tissues are mechanically strong and stiff but can still heal from damage. By contrast, synthetic hydrogels have not shown comparable combinations of properties, as current stiffening approaches inevitably suppress the required chain/bond dynamics for self-healing. Here we show a stiff and self-healing hydrogel with a modulus of 50 MPa and tensile strength up to 4.2 MPa by polymer entanglements in co-planar nanoconfinement. This is realized by polymerizing a highly concentrated monomer solution within a scaffold of fully delaminated synthetic hectorite nanosheets, shear oriented into a macroscopic monodomain. The resultant physical gels show self-healing efficiency up to 100% despite the high modulus, and high adhesion shear strength on a broad range of substrates. This nanoconfinement approach allows the incorporation of novel functionalities by embedding colloidal materials such as MXenes and can be generalized to other polymers and solvents to fabricate stiff and self-healing gels for soft robotics, additive manufacturing and biomedical applications.

The yeast prion protein Sup35 is aggregation-prone at high concentrations. De novo Sup35 prion formation occurs at a significantly increased rate after transient overexpression of Sup35 in the presence of another prion, [PIN+], but it is still a rare event. Recent studies uncovered an additional and seemingly more prevalent role of Sup35: at its physiological level, it undergoes phase separation to form reversible condensates in response to transient stress. Stress-induced reversible Sup35 condensation in the [psi(-)] strain enhances cellular fitness after stress ceases, whereas irreversible Sup35 aggregates in the [PSI+] strain do not confer this advantage. However, how Sup35 overexpression, which could potentially lead to irreversible aggregation, affects its condensation under stress conditions remains unclear. In this study, we used a combinatorial method to examine how different levels of Sup35 overproduction and cellular conditions affect the nature, formation, and physical properties of Sup35 assemblies in yeast cells, as well as their impacts on cellular growth. We observed notable morphological distinctions between irreversible Sup35 aggregates and reversible Sup35 condensates, possibly indicating different formation mechanisms. In addition, Sup35 aggregation caused by a very high overexpression level can strongly inhibit cell growth, diminish the formation of stress-induced condensates when Sup35 is completely aggregated, and impair cellular recovery from stress. Together, this study advances our fundamental understanding of the physical properties and formation mechanism of different Sup35 assemblies and their impacts on cellular growth. We conclude that in vivo studies are sensitive to overexpression and can lead to assembly routes that strongly affect functions. IMPORTANCE The role of condensates in living cells is often studied by overexpression. For understanding their physiological role, this can be problematic. Overexpression can shift cellular functions, thereby changing the system under study, and overexpression can also affect the phase behavior of condensates by shifting the position of the system in the underlying phase diagram. Our detailed study of overexpression of Sup35 in S. cerevisiae shows the interplay between these factors and highlights basic features of intracellular condensation such as the balance between condensation and aggregation as well as how cellular localization and responsiveness depend on protein levels. We also apply super-resolution microscopy to highlight details within the cells.

Here, we explore the molecular level origins of the curious temperature and time dependent assembly phase response of silk-like protein materials. Combining molecular dynamics simulations, CD and FTIR spectroscopy, as well as optical microscopy, we examine the assembly phase response of model engineered tri-block protein constructs with a middle intrinsically disordered region and folded terminal domains. We show that, the assembly phase response over a broad temperature range between 20 and 80 °C arises from strong interprotein interactions. The phase transitions are governed by the interplay of changes in the entropy of the flexible glycine-rich regions and the hydrophobic interactions between the α-helices rich in alanine (Ala). Furthermore, we observe irreversible gelation at high temperatures and during aging (time-induced gelation). Thermal gelation rises via interactions between the Ala-rich regions and subsequent formation of β-sheets that crosslink the protein network. On the other hand, the time-induced gel is formed with no notable secondary structure transitions of the middle block via percolation of the protein, which is sensitive to the dimerizing interactions of the terminal domains. Overall, the significance of this work is that we identify time as a separate design variable from the molecular level characteristics and solution conditions of the silk-like protein gels, and extract assembly guidelines for the gel formation and its characteristics.

There is an increasing understanding that condensation is a crucial intermediate step in the assembly of biological materials and for a multitude of cellular processes. To apply and to understand these mechanisms, in vitro biophysical characterisation techniques are central. The formation and biophysical properties of protein condensates depend on a multitude of factors, such as protein concentration, pH, temperature, salt concentration, and presence of other biomolecules as well as protein purification and storage conditions. Here we show how critical the procedures for preparing protein samples for in vitro studies are. We compare two purification methods of the recombinant spider silk protein CBM-AQ12-CBM and study the effect of background molecules, such as DNA, on the formation and properties of the condensates. We characterize the condensates using aggregation induced emitters (AIEs), coalescence studies, and micropipette aspiration. The condensated sample containing background molecules exhibit a lower threshold concentration for condensate formation accompanied by a lower surface tension and longer coalescence time when compared to the pure protein condensates. Furthermore, the partitioning of small AIEs is enhanced in the presence of background molecules. Our results highlight that the purification method and remaining background molecules strongly affect the biophysical properties of spider silk condensates. Using the acquired knowledge about spider silk protein purification we derive guidelines for reproducible condensate formation that will foster the use of spider silk proteins as adhesives or carriers for biomedical applications.

Phosphate plays a vital role in spider silk spinning and has been utilized in numerous artificial silk spinning attempts to replicate the remarkable mechanical properties of natural silk fiber. Its application in artificial processes has, however, yielded varying outcomes. It is thus necessary to investigate the origins and mechanisms behind these differences. By using recombinant silk protein SC-ADF3 derived from the garden spider Araneus diadematus, here, we describe its conformational changes under various conditions, elucidating the effect of phosphate on SC-ADF3 silk protein properties and interactions. Our results demonstrate that elevated phosphate levels induce the irreversible conformational conversion of SC-ADF3 from random coils to β-sheet structures, leading to decreased protein solubility over time. Furthermore, exposure of SC-ADF3 to phosphate stiffens already formed structures and reduces the ability to form new interactions. Our findings offer insights into the underlying mechanism through which phosphate-induced β-sheet structures in ADF3-related silk proteins impede fiber formation in the subsequent phases. From a broader perspective, our studies emphasize the significance of silk protein conformation for functional material formation, highlighting that the formation of β-sheet structures at the initial stages of protein assembly will affect the outcome of material forming processes.

Azobenzenes are versatile photoswitches that garner interest in applications ranging from photobiology to energy storage. Despite their great potential, transforming azobenzene-based discoveries and proof-of-concept demonstrations from the lab to the market is highly challenging. Herein we give an overview of a journey that started from a discovery of hydroxyazobenzene's humidity sensitive isomerisation kinetics, developed into commercialization efforts of azobenzene-containing thin film sensors for optical monitoring of the relative humidity of air, and arrives to the present work aiming for better design of such sensors by understanding the different factors affecting the humidity sensitivity. Our concept is based on thermal isomerisation kinetics of tautomerizable azobenzenes in polymer matrices which, using pre-defined calibration curves, can be converted to relative humidity at known temperature. We present a small library of tautomerizable azobenzenes exhibiting humidity sensitive isomerisation kinetics in hygroscopic polymer films. We also investigate how water absorption properties of the polymer used, and the isomerisation kinetics are linked and how the azobenzene content in the thin film affects both properties. Based on our findings we propose simple strategies for further development of azobenzene-based optical humidity sensors. Many variables related to azobenzene-based humidity sensors remain. Here, a small library of tautomerisable azobenzenes is studied for their humidity dependent thermal isomerisation rate in polymer thin films.

Gelation of protein condensates formed by liquid-liquid phase separation occurs in a wide range of biological contexts, from the assembly of biomaterials to the formation of fibrillar aggregates, and is therefore of interest for biomedical applications. Soluble-to-gel (sol-gel) transitions are controlled through macroscopic processes such as changes in temperature or buffer composition, resulting in bulk conversion of liquid droplets into microgels within minutes to hours. Using microscopy and mass spectrometry, we show that condensates of an engineered mini-spidroin (NT2repCTYF) undergo a spontaneous sol-gel transition resulting in the loss of exchange of proteins between the soluble and the condensed phase. This feature enables us to specifically trap a silk-domain-tagged target protein in the spidroin microgels. Surprisingly, laser pulses trigger near-instant gelation. By loading the condensates with fluorescent dyes or drugs, we can control the wavelength at which gelation is triggered. Fluorescence microscopy reveals that laser-induced gelation significantly further increases the partitioning of the fluorescent molecules into the condensates. In summary, our findings demonstrate direct control of phase transitions in individual condensates, opening new avenues for functional and structural characterization.

Despite impressive progress in the field, there are still several major bottlenecks in producing fibers from recombinantly produced spider-silk-like proteins to replicate the extraordinary mechanical properties of spider major ampullate silk. The conventional artificial fiber spinning processes rely primarily on organic solvents to coagulate proteins into fibers and require complex post-treatments to obtain fibers with valuable properties. This is due to challenges in obtaining soluble silk proteins, but also because the native silk spinning process leading to the hierarchical organization of the silk proteins is not fully understood and is hard to replicate in a manner applicable to industrial settings. Here, recombinant spider-silk fusion proteins are efficiently produced and processed into as-spun fibers with a toughness modulus of 120 MJ m-3 and extensibility of 255% using solely aqueous solutions. The spider-silk fusion proteins assemble in a manner similar to that reported for native spider silk: they phase separate induced by salting out, followed by alignment and a secondary structure transition triggered by shear forces and dehydration. Finally, the design of the fusion silk proteins enables straightforward functionalization of the fibers under mild all-aqueous conditions via a simple biomolecular click reaction both pre- and post-spinning. A sustainable aqueous wet spinning method enables an efficient production of artificial spider silk fibers which show excellent extensibility and remarkable toughness. A highly specific and efficient biomolecular click reaction is employed to functionalize these fibers under mild conditions, which offers a promising method to construct multi-functional 1D materials. image

Numerous biomolecular shell-forming condensates are reported in cells and bioengineered in vitro. The relationship between the molecular structure of shell-forming condensates and their biophysical properties remains largely unexplored. To fill this gap, we characterize shell-forming condensates of bioengineered spider silk proteins based on Araneus diadematus major ampulla gland silk fibroin 3 (ADF3) using micropipette aspiration. We observe that condensates can burst during aspiration like soap bubbles or polymersomes, demonstrating the formation of a dense protein layer (shell) at the condensate interface. The tendency to burst is more pronounced for condensates formed from proteins with weakly dimerizing terminal blocks. We develop a model to analyse the aspiration and bursting of the condensates, to obtain the surface and bulk viscosity, and to estimate the shell thickness and viscosity. Understanding and controlling the bursting of condensates will open avenues for their use in materials, as compartments for reactions or drug delivery systems. Condensates with a shell can be formed by liquid-liquid phase separation and can burst like viscous bubbles by nucleation and growth of a hole in the shell surrounded by a rim. The authors develop a model to extract a broad range of rheological properties for spherical shells to understand the conditions for bursting.

Recombinant silk proteins provide a route toward sustainable and biocompatible materials. For making such materials, the assembly process from dilute protein into a functional material is central. The assembly mechanism in engineered materials is by necessity different from the natural ones-this poses challenges but also opens opportunities for scaling up and for developing novel properties. The phase behavior of a mini-spidroin, NT-2Rep-CT is studied, which is a widely studied variant of recombinant silk. NT-2Rep-CT can be triggered to assemble by lowering the pH, but even at high pH-considered as storage conditions-it can be in various states, such as forming condensates, clusters, gels, and soluble protein. It is shown how its assembly phases evolve through both metastable and dynamically arrested states. The observed behavior of silk protein solutions is highly complex, and elements thereof from phase diagrams associated with polymers, colloidal systems, and globular proteins are found. Based on the characterization of cluster formation and structural intermediates, a minimalist phase diagram is proposed for NT-2Rep-CT and argues that the understanding and insight into silk assembly via its phase behavior, and especially the arrested states, is central for designing recombinant silk proteins and their processing for materials applications.

Abstract High performance bio‐based materials are an important part of future sustainable technology, and engineered proteins provide excellent possibilities as functional polymers. Adhesives are widely needed for composite materials and biomimetic structures. In biological adhesives, two features have emerged as especially interesting—the role of coacervation and the presence of 3,4‐dihydroxyphenylalanine (DOPA). To study these, protein engineering is used to construct a hybrid silk‐mussel foot protein (mfp) adhesive. Tyr residues in the purified mfp are oxidized to DOPA and an encoded SpyCatcher‐Tag system allowed easy click‐chemistry to couple silk and mfp and to study the parts separately. The combined silk‐mfp protein have a strong tendency to coacervate. DOPA affected the properties of coacervates and increased adhesion by several ways of measuring. In lap shear testing, the combined mfp‐silk protein is superior to any of the components studied separately. Coacervation is suggested to contribute to the adhesion of silk‐mfp, and shows several features suggested to lead to the strength and toughness of natural adhesives. In the lap shear system, coacervation have a stronger overall effect on adhesion than the presence of DOPA. The results show that protein design provides a route toward high performance biosynthetic polymers and future sustainable materials.

Graphical Abstract The wide chemical diversity in bacterial languages is explored in the Research Article by Markus B. Linder et al. (e202314469). The structural similarities were mapped theoretically and sets of both closely related and widely diverging ligand structures experimentally investigated. The results suggest that structural diversity in quorum sensing ligands can be used as a starting point to predict non-cognate binding in intercellular signalling systems. Orthogonal signalling systems could significantly advance our engineering capabilities of microbial communities. Cover designed by Dr. Ekaterina Osmekhina.

For fiber technology, new approaches such as biomimetic materials, such as silks, are being intensively explored, providing new solutions for a variety of industries, including textiles, composites, and biomedical engineering. New approaches for spinning or these materials are needed. Despite recent advances in enhancing fiber tensile properties, achieving significant improvement in tensile properties remains a tedious and challenging task, suffering from little to no controlled extrusion process and difficult optimization in high‐dimensional parameter spaces. Herein, a novel robotic biomimetic pulling method that can rapidly enhance fiber tensile properties surpassing current methods in both speed and resulting fiber properties is shown. Using a controlled fiber‐pulling device with in situ tensile measurements and adaptive optimization based on Bayesian Optimization, fiber strength exceeding 300% of the traditional full factorial design method within just a few experimental iterations is reached. The rapid experimental method presents a potential avenue for enhancing the performance of artificial fibers across diverse industries and applications.

Understanding how native silk spinning occurs is crucial for designing artificial spinning systems. One often overlooked factor in Bombyx mori is the secretion of sericin proteins. Herein, we investigate the variation in amino acid content at different locations in the middle silk gland (MSG) of B. mori. This variation corresponds to an increase in sericin content when moving towards the anterior region of the MSG, while the posterior region predominantly contains fibroin. We estimate the mass ratio of sericin to fibroin to be ~25/75 wt% in the anterior MSG, depending on the fitting method. Then, we demonstrate that the improvement in the extensional behavior of the silk dope in the MSG correlates with the increase in sericin content. The addition of sericin may decrease the viscosity of the silk dope, a factor associated with an increase in the spinnability of silk. We further discuss whether this effect could also result from other known physicochemical changes within the MSG.

Recombinantly produced collagens present a sustainable, ethical, and safe substitute for collagens derived from natural sources. However, controlling the folding of the recombinant collagens, crucial for replicating the mechanical properties of natural materials, remains a formidable task. Collagen-like proteins from willow sawfly are relatively small and contain no hydroxyprolines, presenting an attractive alternative to the large and post-translationally modified mammalian collagens. Utilizing CD spectroscopy and analytical ultracentrifugation, we demonstrate that recombinant willow sawfly collagen assembles into collagen triple helices in a concentration-dependent manner. Interestingly, we observed that the lower concentration threshold for the folding can be overcome by freezing or adding crowding agents. Microscopy data show that both freezing and the addition of crowding agents induce phase separation. We propose that the increase in local protein concentration during phase separation drives the nucleation-step of collagen folding. Finally, we show that freezing also induces the folding of recombinant human collagen fragments and accelerates the folding of natural bovine collagen, indicating the potential to apply phase separation as a universal mechanism to control the folding of recombinant collagens. We anticipate that the results provide a method to induce the nucleation of collagen folding without any requirements for genetic engineering or crosslinking.