Diffusive Dynamic Modes of Recombinant Squid Ring Teeth Proteins by Neutron Spectroscopy.

Stimuli-responsive structural proteins are emerging as promising biocompatible materials for a wide range of biological and nonbiological applications. To understand the physical properties of structural proteins and to replicate their performance in biosynthetic systems, there is a need to understand the molecular mechanisms and relationships that regulate their structure, dynamics, and properties. Here, we study the dynamics of a recombinant squid-inspired protein from Loligo vulgaris (Lv18) by elastic and quasielastic neutron scattering (QENS) to understand the connection between nanostructure, chain dynamics, and mechanical properties. Lv18 is a semicrystalline structural protein, which is plasticized by water above its glass transition temperature at 35 °C. Elastic scans revealed an increased protein chain mobility upon hydration, superimposed dynamic processes, and a decrease in dynamic transition temperatures. Further analysis by QENS revealed that while dry Lv18 protein dynamics are dominated by localized methyl group rotations, hydrated Lv18 dynamics are dominated by the confined diffusion of flexible chains within a ß-sheet nanocrystalline network (8 Å of confinement radius). Our findings establish a relationship between the segment block architecture of Lv18, the diffusive motions within the protein structure, and the mechanical properties of recombinant squid proteins, which will advance the molecular design of novel high-performance protein-inspired materials with tailored dynamics and mechanical properties.

Hydration-Induced Structural Transitions in Biomimetic Tandem Repeat Proteins.

A major challenge in developing biomimetic, high-performance, and sustainable products is the accurate replication of the biological materials' striking properties, such as high strength, self-repair, and stimuli-responsiveness. The rationalization of such features on the microscopic scale, together with the rational design of synthetic materials, is currently hindered by our limited understanding of the sequence-structure-property relationship. Here, employing state-of-the-art nuclear magnetic resonance (NMR) spectroscopy, we link the atomistic structural and dynamic properties of an artificial bioinspired tandem repeat protein TR(1,11) to its stunning macroscopic properties including high elasticity, self-healing capabilities, and record-holding proton conductivity among biological materials. We show that the hydration-induced structural rearrangement of the amorphous Gly-rich soft segment and the ordered Ala-rich hard segment is the key to the material's outstanding physical properties. We found that in the hydrated state both the Ala-rich ordered and Gly-rich disordered parts contribute to the formation of the nanoconfined ß-sheets, thereby enhancing the strength and toughness of the material. This restructuring is accompanied by fast proline ring puckering and backbone cis-trans isomerization at the water-protein interface, which in turn enhances the elasticity and the thermal conductivity of the hydrated films. Our in-depth characterization provides a solid ground for the development of next-generation materials with improved properties.

Biosynthetic self-healing materials for soft machines.

Self-healing materials are indispensable for soft actuators and robots that operate in dynamic and real-world environments, as these machines are vulnerable to mechanical damage. However, current self-healing materials have shortcomings that limit their practical application, such as low healing strength (below a megapascal) and long healing times (hours). Here, we introduce high-strength synthetic proteins that self-heal micro- and macro-scale mechanical damage within a second by local heating. These materials are optimized systematically to improve their hydrogen-bonded nanostructure and network morphology, with programmable healing properties (2-23 MPa strength after 1 s of healing) that surpass by several orders of magnitude those of other natural and synthetic soft materials. Such healing performance creates new opportunities for bioinspired materials design, and addresses current limitations in self-healing materials for soft robotics and personal protective equipment.

Self-Assembly of Topologically Networked Protein-Ti3C2Tx MXene Composites.

Hierarchical organization plays an important role in the stunning physical properties of natural and synthetic composites. Limits on the physical properties of such composites are generally defined by percolation theory and can be systematically altered using the volumetric filler fraction of the inorganic/organic phase. In natural composites, organic materials such as proteins that interact with inorganic filler materials can further alter the hierarchical order and organization of the composite via topological interactions, expanding the limits of the physical properties defined by percolation theory. However, existing polymer systems do not offer a topological parameter that can systematically modulate the assembly characteristics of composites. Here, we present a composite based on proteins and titanium carbide (Ti3C2Tx) MXene that manifests a topological network that regulates the organization, and hence physical properties, of these biomimetic composites. We designed, recombinantly expressed, and purified synthetic proteins consisting of polypeptides with repeating amino acid sequences (tandem repeats) that have the ability to self-assemble into topologically networked biomaterials. We demonstrated that the interlayer distance between MXene sheets can be controlled systematically by the number of tandem repeat units. We varied the filler fraction and number of tandem repeat units to regulate the in-plane and out-of-plane electrical conductivities of these composites. Once Ti3C2Tx MXene sheets are separated enough to facilitate formation of cross-links in our proteins with the number of tandem repeat units reaching 11, the linear I-V characteristics of the composites switched into nonlinear I-V curves with a distinct hysteresis for out-of-plane electron transport, while the in-plane I-V characteristics remained linear. This highlights the impact of synthetic protein templates, which can be designed to modulate electronic transport in composites both isotropically and anisotropically.

Tunable thermal transport and reversible thermal conductivity switching in topologically networked bio-inspired materials.

The dynamic control of thermal transport properties in solids must contend with the fact that phonons are inherently broadband. Thus, efforts to create reversible thermal conductivity switches have resulted in only modest on/off ratios, since only a relatively narrow portion of the phononic spectrum is impacted. Here, we report on the ability to modulate the thermal conductivity of topologically networked materials by nearly a factor of four following hydration, through manipulation of the displacement amplitude of atomic vibrations. By varying the network topology, or crosslinked structure, of squid ring teeth-based bio-polymers through tandem-repetition of DNA sequences, we show that this thermal switching ratio can be directly programmed. This on/off ratio in thermal conductivity switching is over a factor of three larger than the current state-of-the-art thermal switch, offering the possibility of engineering thermally conductive biological materials with dynamic responsivity to heat.

Mechanical Properties of Tandem-Repeat Proteins Are Governed by Network Defects.

Topological defects in highly repetitive structural proteins strongly affect their mechanical properties. However, there are no universal rules for structure-property prediction in structural proteins due to high diversity in their repetitive modules. Here, we studied the mechanical properties of tandem-repeat proteins inspired by squid ring teeth proteins using rheology and tensile experiments as well as spectroscopic and X-ray techniques. We also developed a network model based on entropic elasticity to predict structure-property relationships for these proteins. We demonstrated that shear modulus, elastic modulus, and toughness scale inversely with the number of repeats in these proteins. Through optimization of structural repeats, we obtained highly efficient protein network topologies with 42 MJ/m3 ultimate toughness that are capable of withstanding deformations up to 350% when hydrated. Investigation of topological network defects in structural proteins will improve the prediction of mechanical properties for designing novel protein-based materials.

Ultrafast laser-probing spectroscopy for studying molecular structure of protein aggregates.

We report the development of a new technique to screen protein aggregation based on laser-probing spectroscopy with sub-picosecond resolution. Protein aggregation is an important topic for materials science, fundamental biology as well as clinical studies in neurodegenerative diseases and translation studies in biomaterials engineering. However, techniques to study protein aggregation and assembly are limited to infrared spectroscopy, fluorescent assays, immunostaining, or functional assays among others. Here, we report a new technique to characterize protein structure-property relationship based on ultrafast laser-probing spectroscopy. First, we show theoretically that the temperature dependence of the refractive index of a protein is correlated to its crystallinity. Then, we performed time-domain thermo-transmission experiments on purified semi-crystalline proteins, both native and recombinant (i.e., silk and squid ring teeth), and also on intact E. coli cells bearing overexpressed recombinant protein. Our results demonstrate, for the first time, relative quantification of crystallinity in real time for protein aggregates. Our approach can potentially be used for screening an ultra-large number of proteins in vivo. Using this technique, we could answer many fundamental questions in structural protein research, such as the underlying sequence-structure relationship for protein assembly and aggregation.

Self-Healing Textile: Enzyme Encapsulated Layer-by-Layer Structural Proteins.

Self-healing materials, which enable an autonomous repair response to damage, are highly desirable for the long-term reliability of woven or nonwoven textiles. Polyelectrolyte layer-by-layer (LbL) films are of considerable interest as self-healing coatings due to the mobility of the components comprising the film. In this work mechanically stable self-healing films were fabricated through construction of a polyelectrolyte LbL film containing squid ring teeth (SRT) proteins. SRTs are structural proteins with unique self-healing properties and high elastic modulus in both dry and wet conditions (>2 GPa) due to their semicrystalline architecture. We demonstrate LbL construction of multilayers containing native and recombinant SRT proteins capable of self-healing defects. Additionally, we show these films are capable of utilizing functional biomolecules by incorporating an enzyme into the SRT multilayer. Urease was chosen as a model enzyme of interest to test its activity via fluorescence assay. Successful construction of the SRT films demonstrates the use of mechanically stable self-healing coatings, which can incorporate biomolecules for more complex protective functionalities for advanced functional fabrics.

Molecular tandem repeat strategy for elucidating mechanical properties of high-strength proteins.

Many globular and structural proteins have repetitions in their sequences or structures. However, a clear relationship between these repeats and their contribution to the mechanical properties remains elusive. We propose a new approach for the design and production of synthetic polypeptides that comprise one or more tandem copies of a single unit with distinct amorphous and ordered regions. Our designed sequences are based on a structural protein produced in squid suction cups that has a segmented copolymer structure with amorphous and crystalline domains. We produced segmented polypeptides with varying repeat number, while keeping the lengths and compositions of the amorphous and crystalline regions fixed. We showed that mechanical properties of these synthetic proteins could be tuned by modulating their molecular weights. Specifically, the toughness and extensibility of synthetic polypeptides increase as a function of the number of tandem repeats. This result suggests that the repetitions in native squid proteins could have a genetic advantage for increased toughness and flexibility.

Segmented molecular design of self-healing proteinaceous materials.

Hierarchical assembly of self-healing adhesive proteins creates strong and robust structural and interfacial materials, but understanding of the molecular design and structure-property relationships of structural proteins remains unclear. Elucidating this relationship would allow rational design of next generation genetically engineered self-healing structural proteins. Here we report a general self-healing and -assembly strategy based on a multiphase recombinant protein based material. Segmented structure of the protein shows soft glycine- and tyrosine-rich segments with self-healing capability and hard beta-sheet segments. The soft segments are strongly plasticized by water, lowering the self-healing temperature close to body temperature. The hard segments self-assemble into nanoconfined domains to reinforce the material. The healing strength scales sublinearly with contact time, which associates with diffusion and wetting of autohesion. The finding suggests that recombinant structural proteins from heterologous expression have potential as strong and repairable engineering materials.

Recent advances in nanoscale bioinspired materials.

Natural materials have been a fundamental part of human life since the dawn of civilization. However, due to exploitation of natural resources and cost issues, synthetic materials replaced bio-derived materials in the last century. Recent advances in bio- and nano-technologies pave the way for developing eco-friendly materials that could be produced easily from renewable resources at reduced cost and in a broad array of useful applications. This feature article highlights structural and functional characteristics of bio-derived materials, which will expedite the design fabrication and synthesis of eco-friendly and recyclable advanced nano-materials and devices.

Nanomechanical actuation driven by light-induced DNA fuel.

We report the reversible nanomechanical actuation of a microcantilever driven by the light irradiation-induced conformational changes of i-motif DNA chains, which are functionalized on the cantilever's surface. It is shown that light irradiation-driven nanomechanical actuation can be manipulated using DNA hybridization and/or ionic concentrations.

Preparation and characterization of nanoporous thin films from fully aliphatic polyimides.

Fully aliphatic polyimides (APIs) were prepared from rel-(1'R,3S5'S)-spiro[furan-3(2H),6'-[3]oxabicyclo[3.2.1]octane]-2,2',4',5(4H)-tetrone (DAn) as unsymmetrical spiro dianhydride, and either cis-trans-1,4-diaminocyclohexane (mix-DACH) or trans-1,4-diaminocyclohexane (trans-DACH) as diamine. Structure of all prepared monomers and polymers was confirmed via 1H-NMR and FT-IR. The solubility, optical transparency, and thermal properties of the full APIs were investigated. The solubility and decomposition temperature of the full APIs were found to be correlated with their intermolecular regularity confirmed via wide-angle X-ray diffraction (WAXD). Triblock copolyimides were synthesized through the incorporation of a thermally labile polymer, poly(propylene glycol) (PPG), into the full APIs, and their thermal properties were studied via thermogravimetric analysis (TGA). Nanoporous thin films of the full APIs were prepared via thermolysis of the labile block in the copolyimide films. Phase separation and nanopore formation in the copolymer films were confirmed via atomic force microscopy (AFM) and scanning electron microscopy (SEM), respectively. Nanoporous pores were successfully prepared inside the films.

Single-step electropolymerization patterning of a polypyrrole nanowire by ultra-short pulses via an AFM cantilever.

Conducting polymers (CPs) have attracted a great deal of attention due to their unique properties; these properties are useful in implementing various functional devices, such as memory, and chemical and biological sensors. In particular, the nanopatterning of CPs is a key technology that will accelerate the adoption of CPs in fabricating nanoscaled multifunctional devices. This paper presents an innovative technique for forming polypyrrole nanowire (PPy-NW) patterns, without any additional pretreatment on the gold surface, using atomic force microscopy (AFM) and ultra-short pulse voltage. Applying the ultra-short pulse voltage to the AFM tip has the following advantage: since the electrochemical current is extremely localized around the tip, the successful formation of CP nanowires results. This is because the pulse width is much shorter than the resistor-capacitor (RC) time constant of the equivalent electrochemical circuit of our experimental set-up. This paper provides systematic results regarding the dimensional variation of the PPy-NW patterns produced by varying the electrical conditions of the ultra-short pulse, such as the pulse amplitude, width, and frequency. The results show that use of an ultra-short pulse is essential in fabricating PPy-NW patterns. Additionally, an ultra-short pulse offers excellent pattern controllability for both width (353 nm ∼ 3.37 µm) and height (2.0 ∼ 88.3 nm).

Experimental and numerical study of electrochemical nanomachining using an AFM cantilever tip.

We fabricated nanopatterns on Cu thin films via an electrochemical route using an atomic force microscope (AFM). Experimental results were compared with an equivalent electrochemical circuit model representing an electrochemical nanomachining (ECN) technique. In order to precisely construct the nanopatterns, an ultra-short pulse was applied onto the Cu film through the AFM cantilever tip. The line width of the nanopatterns (the lateral dimension) increased with increased pulse amplitude, on-time, and frequency. The tip velocity effect on the nanopattern line width was also investigated. The study described here provides important insight for fabricating nanopatterns precisely using electrochemical methods with an AFM cantilever tip.

Determination of optimal electrode positions for transcranial direct current stimulation (tDCS).

The present study introduces a new approach to determining optimal electrode positions in transcranial direct current stimulation (tDCS). Electric field and 3D conduction current density were analyzed using 3D finite element method (FEM) formulated for a dc conduction problem. The electrode positions for minimal current injection were optimized by changing the Cartesian coordinate system into the spherical coordinate system and applying the (2+6) evolution strategy (ES) algorithm. Preliminary simulation studies applied to a standard three-layer head model demonstrated that the proposed approach is promising in enhancing the performance of tDCS.