Fine structural analysis of degummed fibroin fibers reveals its superior mechanical capabilities.

Silk fibers constitute a class of protein building blocks capable of functionalization and reprocessing into various material formats. The properties of these fibers are typically affected by the intense thermal treatments needed to remove the sericin gum coating layer. Additionally, their mechanical characteristics are often misinterpreted by assuming the cross-sectional area is a perfect circle. The thermal treatments impact not only the mechanics of the fibers but also the structure of the resolubilized protein, thereby limiting the performance of the resulting silk-based materials. To mitigate these limitations, we explored varying alkali conditions at low temperatures for surface treatment, effectively removing the sericin gum layer while preserving the molecular structure of the fibroin protein, thus, maintaining the hierarchical integrity of the exposed fibroin microfiber core. The precise determination of the initial CSA of the asymmetrical silk fibers led to a comprehensive analysis of their mechanical properties. Our findings indicate that the alkali surface treatment raised the Young's modulus and tensile strength by increasing the fibers' crystallinity by approximately 40% and 50%, respectively, without compromising their strain. We have shown that this treatment facilitated further production of high-purity soluble silk with rheological and self-assembly characteristics comparable to those of native silk.

From Basic Principles of Protein-Polysaccharide Association to the Rational Design of Thermally Sensitive Materials.

Biology resolves design requirements toward functional materials by creating nanostructured composites, where individual components are combined to maximize the macroscale material performance. A major challenge in utilizing such design principles is the trade-off between the preservation of individual component properties and emerging composite functionalities. Here, polysaccharide pectin and silk fibroin were investigated in their composite form with pectin as a thermal-responsive ion conductor and fibroin with exceptional mechanical strength. We show that segregative phase separation occurs upon mixing, and within a limited compositional range, domains ∼50 nm in size are formed and distributed homogeneously so that decent matrix collective properties are established. The composite is characterized by slight conformational changes in the silk domains, sequestering the hydrogen-bonded ß-sheets as well as the emergence of randomized pectin orientations. However, most dominant in the composite's properties is the introduction of dense domain interfaces, leading to increased hydration, surface hydrophilicity, and increased strain of the composite material. Using controlled surface charging in X-ray photoelectron spectroscopy, we further demonstrate Ca ions (Ca2+) diffusion in the pectin domains, with which the fingerprints of interactions at domain interfaces are revealed. Both the thermal response and the electrical conductance were found to be strongly dependent on the degree of composite hydration. Our results provide a fundamental understanding of the role of interfacial interactions and their potential applications in the design of material properties, polysaccharide-protein composites in particular.

Designing Multifunctional Biomaterials via Protein Self-Assembly.

Protein self-assembly is a fundamental biological process where proteins spontaneously organize into complex and functional structures without external direction. This process is crucial for the formation of various biological functionalities. However, when protein self-assembly fails, it can trigger the development of multiple disorders, thus making understanding this phenomenon extremely important. Up until recently, protein self-assembly has been solely linked either to biological function or malfunction; however, in the past decade or two it has also been found to hold promising potential as an alternative route for fabricating materials for biomedical applications. It is therefore necessary and timely to summarize the key aspects of protein self-assembly: how the protein structure and self-assembly conditions (chemical environments, kinetics, and the physicochemical characteristics of protein complexes) can be utilized to design biomaterials. This minireview focuses on the basic concepts of forming supramolecular structures, and the existing routes for modifications. We then compare the applicability of different approaches, including compartmentalization and self-assembly monitoring. Finally, based on the cutting-edge progress made during the last years, we summarize the current knowledge about tailoring a final function by introducing changes in self-assembly and link it to biomaterials' performance.

Protein Compartments Modulate Fibrillar Self-Assembly.

A notable feature of complex cellular environments is protein-rich compartments that are formed via liquid-liquid phase separation. Recent studies have shown that these biomolecular condensates can play both promoting and inhibitory roles in fibrillar protein self-assembly, a process that is linked to Alzheimer's, Parkinson's, Huntington's, and various prion diseases. Yet, the exact regulatory role of these condensates in protein aggregation remains unknown. By employing microfluidics to create artificial protein compartments, the self-assembly behavior of the fibrillar protein lysozyme within them can be characterized. It is observed that the volumetric parameters of protein-rich compartments can change the kinetics of protein self-assembly. Depending on the change in compartment parameters, the lysozyme fibrillation process either accelerated or decelerated. Furthermore, the results confirm that the volumetric parameters govern not only the nucleation and growth phases of the fibrillar aggregates but also affect the crosstalk between the protein-rich and protein-poor phases. The appearance of phase-separated compartments in the vicinity of natively folded protein complexes triggers their abrupt percolation into the compartments' core and further accelerates protein aggregation. Overall, the results of the study shed more light on the complex behavior and functions of protein-rich phases and, importantly, on their interaction with the surrounding environment.

Flexible Soft-Printed Polymer Films with Tunable Plasmonic Properties.

Noble metal nanoparticles (NPs) and particularly gold (Au) have become emerging materials in recent decades due to their exceptional optical properties, such as localized surface plasmons. Although multiple and relatively simple protocols have been developed for AuNP synthesis, the functionalization of solid surfaces composed of soft matter with AuNPs often requires complex and multistep processes. Here we developed a facile approach for functionalizing soft adhesive flexible films with plasmonic AuNPs. The synthetic route is based on preparing Au nanoislands (AuNI) (ca. 2-300 nm) on a glass substrate followed by hydrophobization of the functionalized surface, which in turn, allows efficient transfer of AuNIs to flexible adhesive films via soft-printing tape lithography. Here we show that the AuNI structure remained intact after the hydrophobization and soft-printing procedures. The AuNI-functionalized flexible films were characterized by various techniques, revealing unique characteristics such as tunable localized plasmon resonance and Raman enhancement factors beneficial for chemical and biological sensing applications.

Effects of sound energy on proteins and their complexes.

Mechanical energy in the form of ultrasound and protein complexes intuitively have been considered as two distinct unrelated topics. However, in the past few years, increasingly more attention has been paid to the ability of ultrasound to induce chemical modifications on protein molecules that further change protein-protein interaction and protein self-assembling behavior. Despite efforts to decipher the exact structure and the behavior-modifying effects of ultrasound on proteins, our current understanding of these aspects remains limited. The limitation arises from the complexity of both phenomena. Ultrasound produces multiple chemical, mechanical, and thermal effects in aqueous media. Proteins are dynamic molecules with diverse complexation mechanisms. This review provides an exhaustive analysis of the progress made in better understanding the role of ultrasound in protein complexation. It describes in detail how ultrasound affects an aqueous environment and the impact of each effect separately and when combined with the protein structure and fold, the protein-protein interaction, and finally the protein self-assembly. It specifically focuses on modifying role of ultrasound in amyloid self-assembly, where the latter is associated with multiple neurodegenerative disorders.

Sound-mediated nucleation and growth of amyloid fibrils.

Mechanical energy, specifically in the form of ultrasound, can induce pressure variations and temperature fluctuations when applied to an aqueous media. These conditions can both positively and negatively affect protein complexes, influencing their stability, folding patterns, and self-assembling behavior. Regarding understanding the effects of ultrasound on the self-assembly of amyloidogenic proteins, our knowledge remains quite limited. In our recent work, we established the boundary conditions under which sound energy can either cause damage or induce only negligible changes in the structure of protein species. In the present study, we demonstrate that when the delivered ultrasonic energy is sufficiently low, it can induce refolding of specific motifs in protein monomers, as it has been revealed by MD, which is sufficient for primary nucleation, characterized by adopting a hydrogen-bonded ß -sheet-rich structure. These structural changes are initiated by pressure perturbations and are accelerated by a temperature factor. Furthermore, the prolonged action of low-amplitude ultrasound enables the elongation of amyloid protein nanofibrils directly from monomeric lysozyme proteins, in a controlled manner, until they reach a critical length. Using solution X-ray scattering, we determined that nanofibrillar assemblies, formed under the influence of ultrasound energy and natively fibrillated lysozyme, share identical structural characteristics. Thus, these results contribute to our understanding of the effects of ultrasound on fibrillar protein self-assembly and lay the foundation for the potential exploitation of sound energy in a protein chemistry environment.

Microcompartmentalization Controls Silk Feedstock Rheology.

The rheological characteristics of pre-spun native silk protein, which is stored as a viscous pulp inside the silk gland, are the key factors that determine the mechanical performance of the endpoint material: the spun silk fibers. In silkworms and arthropods, microcompartmentalization was shown to play an important regulatory role in storing and stabilizing the aggregation-prone silk and in initiating the fibrillar self-assembly process. However, our current understanding of the mechanism of stabilization of the highly unstable protein pulp in its soluble state inside the microcompartments and of the conditions required for initiating the structural transition in protein inside the microcompartments remains limited. Here, we exploited the power of droplet microfluidics to mimic the silk protein's microcompartmentalization event; we introduced changes in the chemical environment and analyzed the storage-to-spinning transition as well as the accompanying structural changes in silk fibroin protein, from its native fold into an aggregative ß-sheet-rich structure. Through a combination of experimental and computational simulations, we established the conditions under which the structural transition in microcompartmentalized silk protein is initiated, which, in turn, is reflected in changes in the silk-rich fluid behavior. Overall, our study sheds light on the role of the independent parameters of a dynamically changing chemical environment, changes in fluid viscosity, and the shear forces that act to balance silk protein self-assembly, and thus, facilitate new exploratory avenues in the field of biomaterials.

Modulating amyloids' formation path with sound energy.

Protein folding is crucial for biological activity. Proteins' failure to fold correctly underlies various pathological processes, including amyloidosis, the aggregation of insoluble proteins (e.g., lysozymes) in organs. The exact conditions that trigger the structural transition of amyloids into ß-sheet-rich aggregates are poorly understood, as is the case for the amyloidogenic self-assembly pathway. Ultrasound is routinely used to destabilize a protein's structure and enhance amyloid growth. Here, we report on an unexpected ultrasound effect on lysozyme amyloid species at different stages of aggregation: ultrasound-induced structural perturbation gives rise to nonamyloidogenic folds. Our infrared and X-ray analyses of the chemical, mechanical, and thermal effects of sound on lysozyme's structure found, in addition to the expected ultrasound-induced damage, evidence of irreversible disruption of the ß-sheet fold of fibrillar lysozyme resulting in their structural transformation into monomers with no ß-sheets. This structural transition is reflected in changes in the kinetics of protein self-assembly, namely, either prolonged nucleation or accelerated fibril growth. Using solution X-ray scattering, we determined the structure, the mass fraction of lysozyme monomer, and the morphology of its filamentous assemblies formed under different sound parameters. A nanomechanical analysis of ultrasound-modified protein assemblies revealed a correlation between the ß-sheet content and elastic modulus of the protein material. Suppressing one of the ultrasound-derived effects allowed us to control the structural transformations of lysozyme. Overall, our comprehensive investigation establishes the boundary conditions under which ultrasound damages protein structure and fold. This knowledge can be utilized to impose medically desirable structural modifications on amyloid ß-sheet-rich proteins.

Dataset for the Synthesis of Boron-Dipyrrin Dyes, their fluorescent properties, their interaction with proteins, Triton-X-based surfactants, and micellar clusterization approaches to validation based on fluorescent dyes.

The data presented here refer to the research article by Aleksei V. Solomonov, Yuriy S. Marfin, Alexander B. Tesler, Dmitry A. Merkushev, Elizaveta A. Bogatyreva, Elena V. Antina, Evgeniy V. Rumyantsev, and Ulyana Shimanovich "Spanning BODIPY fluorescence with self-assembled micellar clusters", Colloids and Surfaces B: Biointerfaces, 216, 2022, 112532. The present article provides details on optical characterization for a set of meso- and tetra-substituted boron-dipyrrin (BODIPY) complexes encapsulated inside of self-assembled Triton-X-based micellar coordination clusters (MCCs), based on Triton-X family surfactants. Changes in the optical properties of the BODIPY complexes upon interaction with bovine serum albumin, in a binary mixture of THF:H2O and titrated with Triton TX-114, were evaluated. The optical properties and the formation kinetics of the BODIPY-based MCCs and the BODIPY-supported micelle chelator aggregates (MCAs) are presented as well. The presented data provide additional insights into the structural and formation aspects of both the traditional and newly obtained micellar coordination clusters for their future optimization, control, and application. The synthetic procedures for the synthesis of a set of meso- and tetra-substituted BODIPY complexes and their optical properties in different media are also presented. The research is related to the paper (Solomonov et al., 2022).

Spanning BODIPY fluorescence with self-assembled micellar clusters.

BODIPY dyes possess favorable optical properties for a variety of applications including in vivo and in vitro diagnostics. However, their utilization might be limited by their water insolubility and incompatibility with chemical modifications, resulting in low aggregation stability. Here, we outline the route for addressing this issue. We have demonstrated two approaches, based on dye entrapment in micellar coordination clusters (MCCs); this provides a general solution for water solubility as well as aggregation stability of the seven BODIPY derivatives. These derivatives have various bulky aromatic substituents in the 2,3,5,6- and meso-positions and can rotate relative to a dipyrrin core, which also provides molecular rotor properties. The molecular structural features and the presence of aromatic groups allows BODIPY dyes to be used as "supporting molecules", thus promoting micelle-micelle interaction and micellar network stabilization. In the second approach, self-micellization, following BODIPY use, leads to MCC formation without the use of any mediators, including chelators and/or metal ions. In both approaches, BODIPY exhibits an excellent optical response, at a concentration beyond its solubilization limit in aqueous media and without undesired crystallization. The suggested approaches represent systems used to encapsulate BODIPY in a capsule-based surfactant environment, enabling one to track the aggregation of BODIPY; these approaches represent an alternative system to study and apply BODIPY's molecular rotor properties. The stabilized compounds, i.e., the BODIPY-loaded MCCs, provide a unique feature of permeability to hydrophilic ligand-switching proteins such as BSA; they exhibit a bright "turn-on" fluorescence signal within the clusters via macromolecular complexation, thus expanding the possibilities of water-soluble BODIPY-loaded MCCs utilization for functional indicators.

Polymer Gel with Tunable Conductive Properties: A Material for Thermal Energy Harvesting.

The spontaneous gelation of poly(4-vinyl pyridine)/pyridine solution produces materials with conductive properties that are suitable for various energy conversion technologies. The gel is a thermoelectric material with a conductivity of 2.2-5.0 × 10-6 S m-1 and dielectric constant ε = 11.3. On the molecular scale, the gel contains various types of hydrogen bonding, which are formed via self-protonation of the pyridine side chains. Our measurements and calculations revealed that the gelation process produces bias-dependent polymer complexes: quasi-symmetric, strongly hydrogen-bonded species, and weakly bound protonated structures. Under an applied DC bias, the gelled complexes differ in their capacitance/conductive characteristics. In this work, we exploited the bias-responsive characteristics of poly(4-vinyl pyridine) gelled complexes to develop a prototype of a thermal energy harvesting device. The measured device efficiency is S = ΔV/ΔT = 0.18 mV/K within the temperature range of 296-360 K. Investigation of the mechanism underlying the conversion of thermal energy into electric charge showed that the heat-controlled proton diffusion (the Soret effect) produces thermogalvanic redox reactions of hydrogen ions on the anode. The charge can be stored in an external capacitor for heat energy harvesting. These results advance our understanding of the molecular mechanisms underlying thermal energy conversion in the poly(4-vinyl pyridine)/pyridine gel. A device prototype, enabling thermal energy harvesting, successfully demonstrates a simple path toward the development of inexpensive, low-energy thermoelectric generators.

Light-Induced Reactions within Poly(4-vinyl pyridine)/Pyridine Gels: The 1,6-Polyazaacetylene Oligomers Formation.

Cyclic 6-membered aromatic compounds such as benzene and azabenzenes (pyridine, pyridazine, and pyrazine) are known to be light-sensitive, affording, in particular, the Dewar benzene type of intermediates. Pyridine is known to provide the only Dewar pyridine intermediate that undergoes reversible ring-opening. We found that irradiation of photosensitive gels prepared from poly(4-vinyl pyridine) and pyridine at 254 or 312 nm leads to pyridine ring-opening and subsequent formation of 5-amino-2,4-pentadienals. We show that this light-induced process is only partially reversible, and that the photogenerated aminoaldehyde and aminoaldehyde-pending groups undergo self-condensation to produce cross-linked, conjugated oligomers that absorb light in the visible spectrum up to the near-infrared range. Such a sequence of chemical reactions results in the formation of gel with two distinct morphologies: spheres and fiber-like matrices. To gain deeper insight into this process, we prepared poly(4-vinyl pyridine) with low molecular weight (about 2000 g/mol) and monitored the respective changes in absorption, fluorescence, 1H-NMR spectra, and electrical conductivity. The conductivity of the polymer gel upon irradiation changes from ionic to electronic, indicative of a conjugated molecular wire behavior. Quantum mechanical calculations confirmed the feasibility of the proposed polycondensation process. This new polyacetylene analog has potential in thermal energy-harvesting and sensor applications.

pH-Responsive Capsules with a Fibril Scaffold Shell Assembled from an Amyloidogenic Peptide.

Peptides and proteins have evolved to self-assemble into supramolecular entities through a set of non-covalent interactions. Such structures and materials provide the functional basis of life. Crucially, biomolecular assembly processes can be highly sensitive to and modulated by environmental conditions, including temperature, light, ionic strength and pH, providing the inspiration for the development of new classes of responsive functional materials based on peptide building blocks. Here, it is shown that the stimuli-responsive assembly of amyloidogenic peptide can be used as the basis of environmentally responsive microcapsules which exhibit release characteristics triggered by a change in pH. The microcapsules are biocompatible and biodegradable and may act as vehicles for controlled release of a wide range of biomolecules. Cryo-SEM images reveal the formation of a fibrillar network of the capsule interior with discrete compartments in which cargo molecules can be stored. In addition, the reversible formation of these microcapsules by modulating the solution pH is investigated and their potential application for the controlled release of encapsulated cargo molecules, including antibodies, is shown. These results suggest that the approach described here represents a promising venue for generating pH-responsive functional peptide-based materials for a wide range of potential applications for molecular encapsulation, storage, and release.

Protein nanofibril design via manipulation of hydrogen bonds.

The process of amyloid nanofibril formation has broad implications including the generation of the strongest natural materials, namely silk fibers, and their major contribution to the progression of many degenerative diseases. The key question that remains unanswered is whether the amyloidogenic nature, which includes the characteristic H-bonded ß-sheet structure and physical characteristics of protein assemblies, can be modified via controlled intervention of the molecular interactions. Here we show that tailored changes in molecular interactions, specifically in the H-bonded network, do not affect the nature of amyloidogenic fibrillation, and even have minimal effect on the initial nucleation events of self-assembly. However, they do trigger changes in networks at a higher hierarchical level, namely enhanced 2D packaging which is rationalized by the 3D hierarchy of ß-sheet assembly, leading to variations in fibril morphology, structural composition and, remarkably, nanomechanical properties. These results pave the way to a better understanding of the role of molecular interactions in sculpting the structural and physical properties of protein supramolecular constructs.

Protein Microgels from Amyloid Fibril Networks.

Nanofibrillar forms of amyloidogenic proteins were initially discovered in the context of protein misfolding and disease but have more recently been found at the origin of key biological functionality in many naturally occurring functional materials, such as adhesives and biofilm coatings. Their physiological roles in nature reflect their great strength and stability, which has led to the exploration of their use as the basis of artificial protein-based functional materials. Particularly for biomedical applications, they represent attractive building blocks for the development of, for instance, drug carrier agents due to their inherent biocompatibility and biodegradability. Furthermore, the propensity of proteins to self-assemble into amyloid fibrils can be exploited under microconfinement, afforded by droplet microfluidic techniques. This approach allows the generation of multi-scale functional microgels that can host biological additives and can be designed to incorporate additional functionality, such as to aid targeted drug delivery.

Fabrication and Characterization of Reconstituted Silk Microgels for the Storage and Release of Small Molecules.

Silk fibroin is a natural protein obtained from the Bombyx mori silkworm. In addition to being the key structural component in silkworm cocoons, it also has the propensity to self-assemble in vitro into hierarchical structures with desirable properties such as high levels of mechanical strength and robustness. Furthermore, it is an appealing biopolymer due to its biocompatability, low immunogenicity, and lack of toxicity, making it a prime candidate for biomedical material applications. Here, it is demonstrated that nanofibrils formed by reconstituted silk fibroin can be engineered into supramolecular microgels using a soft lithography-based microfluidic approach. Building on these results, a potential application for these protein microgels to encapsulate and release small molecules in a controlled manner is illustrated. Taken together, these results suggest that the tailored self-assembly of biocompatible and biodegradable silk nanofibrils can be used to generate functional micromaterials for a range of potential applications in the biomedical and pharmaceutical fields.

Fibrous Protein Self-Assembly in Biomimetic Materials.

Protein self-assembly processes, by which polypeptides interact and independently form multimeric structures, lead to a wide array of different endpoints. Structures formed range from highly ordered molecular crystals to amorphous aggregates. Order arises in the system from a balance between many low-energy processes occurring due to a set of interactions between residues in a chain, between residues in different chains, and between solute and solvent. In Nature, self-assembling protein systems have evolved over millions of years to organize into supramolecular structures, optimized for specific functions, with this propensity determined by the sequence of their constituent amino acids, of which only 20 are encoded in DNA. The structural materials that arise from biological self-assembly can display remarkable mechanical properties, often as a result of hierarchical structure on the nano- and microscales, and much research has been devoted to mimicking and exploiting these properties for a variety of end uses. This work presents a review of a range of studies in which biological functions are effectively reproduced through the design of self-assembling fibrous protein systems.

Biophotonics of Native Silk Fibrils.

Native silk fibroin (NSF) is a unique biomaterial with extraordinary mechanical and biochemical properties. These key characteristics are directly associated with the physical transformation of unstructured, soluble NSF into highly organized nano- and microscale fibrils rich in ß-sheet content. Here, it is shown that this NSF fibrillation process is accompanied by the development of intrinsic fluorescence in the visible range, upon near-UV excitation, a phenomenon that has not been investigated in detail to date. Here, the optical and fluorescence characteristics of NSF fibrils are probed and a route for potential applications in the field of self-assembled optically active biomaterials and systems is explored. In particular, it is demonstrated that NSF can be structured into autofluorescent microcapsules with a controllable level of ß-sheet content and fluorescence properties. Furthermore, a facile and efficient fabrication route that permits arbitrary patterns of NSF microcapsules to be deposited on substrates under ambient conditions is shown. The resulting fluorescent NSF patterns display a high level of photostability. These results demonstrate the potential of using native silk as a new class of biocompatible photonic material.

Sequential Release of Proteins from Structured Multishell Microcapsules.

In nature, a wide range of functional materials is based on proteins. Increasing attention is also turning to the use of proteins as artificial biomaterials in the form of films, gels, particles, and fibrils that offer great potential for applications in areas ranging from molecular medicine to materials science. To date, however, most such applications have been limited to single component materials despite the fact that their natural analogues are composed of multiple types of proteins with a variety of functionalities that are coassembled in a highly organized manner on the micrometer scale, a process that is currently challenging to achieve in the laboratory. Here, we demonstrate the fabrication of multicomponent protein microcapsules where the different components are positioned in a controlled manner. We use molecular self-assembly to generate multicomponent structures on the nanometer scale and droplet microfluidics to bring together the different components on the micrometer scale. Using this approach, we synthesize a wide range of multiprotein microcapsules containing three well-characterized proteins: glucagon, insulin, and lysozyme. The localization of each protein component in multishell microcapsules has been detected by labeling protein molecules with different fluorophores, and the final three-dimensional microcapsule structure has been resolved by using confocal microscopy together with image analysis techniques. In addition, we show that these structures can be used to tailor the release of such functional proteins in a sequential manner. Moreover, our observations demonstrate that the protein release mechanism from multishell capsules is driven by the kinetic control of mass transport of the cargo and by the dissolution of the shells. The ability to generate artificial materials that incorporate a variety of different proteins with distinct functionalities increases the breadth of the potential applications of artificial protein-based materials and provides opportunities to design more refined functional protein delivery systems.