Proteinaceous Fibers with Outstanding Mechanical Properties Manipulated by Supramolecular Interactions

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 Jun 2021Proteinaceous Fibers with Outstanding Mechanical Properties Manipulated by Supramolecular Interactions Jing Sun, Bo Li, Fan Wang, Jing Feng, Chao Ma, Kai Liu and Hongjie Zhang Jing Sun Department of Chemistry, Tsinghua University, Beijing 100084 Institute of Organic Chemistry I, University of Ulm, Ulm 89081 , Bo Li State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 , Fan Wang State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 , Jing Feng State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 , Chao Ma State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 , Kai Liu *Corresponding author: E-mail Address: [email protected] E-mail Address: [email protected] Department of Chemistry, Tsinghua University, Beijing 100084 State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 and Hongjie Zhang Department of Chemistry, Tsinghua University, Beijing 100084 State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 https://doi.org/10.31635/ccschem.020.202000231 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail Proteinaceous fibers based on spidroins have attracted widespread attention due to their lightweight and mechanically strong properties. Presently, mechanical modulation is mainly dependent on the ultrahigh molecular weight of recombinant proteins. This makes it difficult to construct and express the target proteins. It is thus significant to develop alternative strategies for the fabrication of robust biological fibers. Herein, we demonstrate one new type of engineered protein fibers using electrostatic complexation of the cationic elastins and anionic dihydroxyphenylalanine surfactants. Interestingly, the mechanical performance of the resulting fibers can be modulated by multiple supramolecular interactions in the system including electrostatic force, hydrogen bonding, metal coordination, cation–π and other aromatic interactions. Consequently, significant alternation of the fibers' breaking strength (from 32 to 160 MPa), Young's modulus (from 0.8 to 17 GPa), and toughness (from 1.2 to 99 MJ·m−3) has been achieved. Moreover, the fibers exhibit high plasticity; for example, the formation of different helical structures, and strong fluorescence after the introduction of Tb chelation. Therefore, this study offers new strategies for the mechanical regulation of engineered protein fibers. Download figure Download PowerPoint Introduction Lightweight spider silks have been investigated extensively due to their combination of high strength and toughness,1–6 which offers great potential in high-tech applications.7–10 Inspired by this, many attempts have been made to fabricate robust biological fibers, such as regenerated silk fibers10–15 and recombinant protein fibers.16–21 In these studies, harsh reaction conditions were used to fabricate the protein fibers, which may lead to incomplete folding structures and destroyed intermolecular interactions. Moreover, with the expression of the structural proteins by recombinant methods, it is difficult to produce the macromolecules with high molecular weights. Consequently, the as-spun fibers exhibit weak mechanical performance in comparison with native types.22–28 These drawbacks seriously restrict the commercial applications of as-spun fibers. Thus, it is important to develop other effective strategies to improve the mechanical properties of protein fibers. The regulation of supramolecular interactions of macromolecules and control of their structure and orientation play an important role in determining materials' performance. Recent investigations on 3,4-dihydroxyphenylalanine (DOPA)-based materials suggest that the multifunctional nature of the catechol moiety in DOPA plays a crucial role in increasing mechanical strength via its hydrogen bonding, oxidation, or the coordination with metal ions.29–36 By modifying the DOPA component, it is also possible to introduce other supramolecular interactions including electrostatic forces and π–π or cation–π intermolecular interactions.37–42 However, the fabrication of robust biological fibers by the introduction of DOPA is rarely reported. In this context, by a combination of those supramolecular interactions,43–46 it is possible to manipulate the mechanical properties of protein fibers in a flexible way. Therefore, it becomes an attractive goal to design and fabricate mechanically strong protein fibers by utilizing a DOPA-based strategy. Herein, we developed a simple method to produce protein fibers by integrating DOPA-based surfactants (DSS) with positively charged elastin-like polypeptides (ELPs) via electrostatic interaction. The mechanical performance of the resulting protein fibers can be controlled by regulating the supramolecular interaction in the system. It was found that the toughness and stiffness of fibers were modulated from 18 to 99 MJ·m−3 and 0.8 to 8.8 GPa, respectively. Moreover, the fibers exhibited stronger performance after metal ion treatment. For instance, Young's modulus reached 17 GPa due to Fe3+–DOPA chelation, which is superior to artificial spider silks and comparable with some natural ones. This strategy represents a novel concept for exploring bio-inspired, mechanically strong protein materials. Experimental Methods 3,4-Dihydroxylphenyl propionic acid, K2CO3, benzyl bromide, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP), 6-amino-1-hexanol, SO3·NMe3, ion-exchanged resin (Na+ form), and 10% Palladium (Pd) on activated carbon were acquired from Acros (The Netherlands) or Sigma-Aldrich (St. Louis, MO) and used without further purification. All biochemicals for cloning and ELP expression, such as Lysogeny broth medium, salts, antibiotics as well as inducer compounds, were used as received (from Sigma-Aldrich) without any further purification. The pUC19 cloning vector, restriction enzymes, and GeneJET Plasmid Miniprep kit were purchased from Thermo Fisher Scientific (Waltham, MA). Digested DNA fragments were purified using QIAquick spin miniprep kits from QIAGEN (Valencia, CA). Escherichia coli (E. coli) XL1-Blue competent cells for plasmid amplification were purchased from Stratagene (La Jolla, CA). Oligonucleotides for sequencing were ordered from Sigma-Aldrich. For all experiments, ultrapure water (18.2 MΩ) purified by a MilliQ-Millipore system (Millipore, Germany) was used. All other solvents used in this study were analytical grade. Proton nuclear magnetic resonance (1H NMR) and carbon nuclear magnetic resonance (13C NMR) spectra were recorded on a Varian Mercury NMR spectrometer (400 MHz, USA). Chemical shifts (δ) are quoted in parts per million (ppm). All UV–Vis spectra were measured on a JASCO V-630 UV-Vis spectrophotometer (JASCO Benelux B. V., The Netherlands) at 25 °C using 1 mL cuvettes. Samples were dissolved in an appropriate solvent and measured under the same solvent. Data analysis was carried out using Origin 9.0 (OriginLab Corporation, USA). The surface morphology and cross section was measured on a JSM 6320F (JEOL company, The Netherlands) scanning electron microscopy (SEM). Thermogravimetric analysis (TGA) was carried out using a TA Instruments Q1000 (TA instrument, USA) system in a nitrogen atmosphere and with a heating–cooling rate of 20 °C·min−1. Tensile strength was measured on INSTRON 5565 (INSTRON, Germany) at a speed of 10 mm·min−1. Polarized optical microscopy (POM) was conducted on a Zeiss Axiophot (Zeiss company, Germany). Small-angle X-ray scattering (SAXS) was performed by employing a conventional X-ray source with a radiation wavelength of 1.54 Å, and a Bruker Nano/Microstar machine (Bruker company, Germany) was used to obtain small-angle scattering profiles, where the sample-to-detector distance was 24 cm. The sample holder is a metal plate with a small hole (diameter 0.25 cm; thickness 0.15 cm), where the X-ray beam passes through. The elastin-like polypeptides (ELP)-DSS fiber was fixed to the sample holder with tape. The scattering vector q is defined as q = 4π sin θ/λ with 2θ being the scattering angle. More detailed experimental and computational data are available in Supporting Information. Results and Discussion To study the mechanical behaviors of protein fibers, we prepared novel elastin-like polypeptide (ELP)-based fibers by electrostatic complexation between a positively charged ELP backbone and an anionic catechol-based surfactant (DSS) (Figure 1a). The positively charged elastin-like polypeptides consisted of the dominating repetitive pentapeptide sequence (VPG XG)n. Recombinant DNA technology and expression in E. coli installed lysine in position four, X. 47,48 Different chain lengths of ELPs were produced, including K18, K72, and K108, where the number is positive charges per ELP molecule ( Supporting Information Figure S1). The surfactant, DSS (Mn = 383.39 g·mol−1), was synthesized from 3-(3,4-dihydroxyphenyl) propanoic acid by benzyl protection, amidation, sulfonation with SO3·NMe3, followed by deprotection ( Supporting Information Scheme S1). All the compounds were characterized by NMR spectroscopy and high-resolution mass spectroscopy (HRMS). Figure 1 | Schematic illustration of the ELP-DSS fiber formation. (a) The construction and expression of the positively charged ELP by recombinant DNA technology and expression in E. coli. Positively charged ELPs used in this study: K72 and K108. After complexing with DOPA-based surfactant (DSS), centrifuging, lyophilizing, the ELP-DSS fibers can be generated by manual spinning. Photograph for the ELP-K108-DSS fiber. Scale bar is 7 mm. (b) Polarized optical microscopy analysis of the ELP-DSS fiber (here ELP-K108-DSS fiber was measured). The birefringence properties suggest an ordered structure in the network of ELP-K108-DSS fiber. Scale bar is 50 μm. (c) SAXS analysis of the ELP-DSS fiber (here ELP-K108-DSS fiber was measured). The average distance of the formed ELP-K108-DSS complex is approximately 1.9 nm. The inset represents the molecular packing model of ELP-DSS fibers (ELPs are represented in red, surfactant groups are represented in blue). (d) SEM analysis of ELP-DSS fibers showed a fibrillary structure and uniform surface morphology (here ELP-K108-DSS fiber was measured). Download figure Download PowerPoint The ELP-DSS complexes form via electrostatic interaction between positively charged ELPs and anionic DSS surfactant. In general, an aqueous solution of ELP with DSS (1∶1 molar ratio of lysine to surfactant) was mixed to form the ELP-DSS complex. Subsequent dehydration and manual spinning affords the complex (preparation details in Supporting Information). During the ELP-K72-DSS generation, the gel network exhibited rapid water evaporation. But in the ELP-K108-DSS system, the increased chain length led to a gel network with a high capacity for water storage, which makes it suitable for the study of water content on fibers' mechanical performance, as discussed below. To quantitatively evaluate the ELP-DSS complexes, 1H NMR spectroscopy was performed with ELP-K18-DSS ( Supporting Information Figure S2). The stoichiometry of K18 and DSS was measured as 1∶16.7 (i.e., 0.9 DSS molecules per positively charged lysine of the ELP), indicating that approximately 10% of the lysine moieties was not complexed with the surfactant molecules. This might indicates that cation–π interactions of the free lysine moieties in ELP and the phenyl rings in DSS exist in the fiber system. Furthermore, as shown in the UV–Vis spectrum ( Supporting Information Figure S3), there is an obvious absorption at approximately 550 nm for the ELP-K108-DSS complex after Fe3+ ion treatment, indicating the formation of bis Fe-catecholate.39 To gain more insight into ELP fibers, POM analysis revealed significant birefringence under cross-polarized light illumination, indicating an ordered arrangement of molecules in the solid state of the protein fiber (Figure 1b). Further characterization of ELP fibers by SAXS showed profiles with a weak, broad diffraction peak at q = 3.26 nm−1 (Figure 1c). The weak, broad diffraction peak corresponds to the d spacing of approximately 1.9 nm (d = 2π/q), which was attributed to the average diameter of the ELP-DSS complex. In addition, SEM analysis revealed that the fibers have distinctively fibrillary structural features (Figure 1d). Next, the mechanical properties of ELP-K72-DSS fibers were evaluated by uniaxial tensile testing. The typical stress–strain curves are shown in Supporting Information Figure S4. In the absence of metal ions, the tensile strength, Young's modulus, extensibility, and toughness of the fiber are 38 ± 3 MPa, 2.7 ± 0.3 GPa, 2.1 ± 0.9%, and 0.6 ± 0.3 MJ·m−3, respectively ( Supporting Information Table S1). To further reinforce the mechanical behavior of ELP-K72-DSS fibers, different metal ions (Fe3+ or Tb3+) were incorporated. For example, after Fe3+ ion treatment, the water content, confirmed by TGA, of the as-spun fiber was approximately 13% ( Supporting Information Figure S5). Because water evaporated quickly from ELP-K72-DSS fibers, we ascribed this measurement to water within the fibers. The tensile strength and Young's modulus increased dramatically to approximately 66 MPa and 7.8 GPa, respectively (Figures 2a and 2b). While the extensibility and toughness exhibited a trivial loss of approximately 1.0% and 0.5 MJ·m−3, respectively (Figures 2c and 2d). Notably, the high strength and high modulus can be attributed to the metal–DOPA chelation interaction in the fiber. Meanwhile, the intermolecular motion might be restricted by such a chelation interaction, which led to low strain of the fiber. A similar trend for the mechanical performance of ELP-K72-DSS fiber after Tb3+ ion treatment was observed (Figures 2a–2d and Supporting Information Table S1). In addition, SEM analysis showed that the fiber has a uniform cylindrical fibrillary structure and surface morphology after metal ion treatment (Figures 2e and 2f). Remarkably, fiber treated with Tb3+ ions displayed smoother morphology, a more intact cross section, and higher moduli of 9.5 GPa when compared with Fe3+ treated fibers. The enhanced mechanical behaviors can be ascribed to the formation of metal-coordination-chelation bonds in the fiber after metal ion treatment. The rough morphology in the ELP-K72-DSS-Fe fibers might be related to oxidization induced by Fe3+. Figure 2 | Investigation of metal coordination effect on mechanical properties of the ELP-DSS fibers; here ELP-K72-DSS was used as a representative example. (a–d) Mechanical performance analysis of the fibers including (a) breaking strength, (b) Young's Modulus, (c) extensibility, and (d) toughness. All error bars represent standard deviation (n = 3). (e and f) SEM was used to investigate the surface morphology and cross section of the broken ELP fibers. (e) SEM image for the ELP-K72-DSS-Fe fiber. (f) SEM image for the ELP-K72-DSS-Tb fiber. The scale bar is 10 μm. Download figure Download PowerPoint Subsequently, the ELP with K108 was used to fabricate protein fibers under the same conditions. As shown in Figure 3, the mechanical performances of ELP-K108-DSS fibers were significantly affected by the water content and the supramolecular interactions in the fiber. In a combination of the tensile test (Figures 3a–3d) and TGA ( Supporting Information Figure S6), at high water content of approximately 45%, the obtained tensile strength and Young's modulus were 32 ± 0.3 MPa and 0.8 ± 0.1 GPa, respectively (Figure 3e). Particularly, the extensibility and the toughness of ELP-K108-DSS fibers reached 332 ± 24% and 99 ± 19 MJ·m−3, respectively ( Supporting Information Table S2), which is comparable, or even higher, than that of many recombinant spider silks or regenerated silkworm fibers. In addition, the exceptional extensibility endows ELP-K108-DSS fibers with an excellent macroscopic resilience property even when manually stretched 14 times its initial size ( Supporting Information Movie S1). Furthermore, the mechanical performance of ELP-K108-DSS fibers varied as water content fluctuated (Figure 3f). Accordingly, the recorded tensile strength and Young's modulus increased to 55 ± 16 MPa and 8.9 ± 2.1 GPa, respectively, due to decreased water content (∼11%) ( Supporting Information Figure S7). Meanwhile, the extensibility and toughness dropped dramatically to 60 ± 26% and 18 ± 8 MJ·m−3, respectively ( Supporting Information Table S2). These behaviors indicated that the mechanical performance of ELP-K108-DSS fibers was highly dependent on water content because of the water molecules acting as plasticizers within the fibers. Figure 3 | Mechanical characterization of the ELP-DSS fibers under different preparation conditions. (a–d) Mechanical performance analysis of the fibers including (a) breaking strength, (b) Young's Modulus, (c) extensibility, and (d) toughness. All error bars represent standard deviation (n = 3). The ELP-K108-DSS fibers were used as a representative example. (e–g) Typical stress–strain curves of the ELP-DSS fibers under different preparation conditions. (e) The ELP-K108-DSS fiber containing 45% water content and the inset is the magnified part in the blue-dotted area. (f) The ELP-K108-DSS fiber containing 11% water content, (g) The ELP-K108-DSS fiber after Fe3+ ion treatment. (h) Schematic for the possible molecular mechanism in the mechanical modulation of ELP-DSS fibers. Color codes represent each supramolecular interaction: gray (electrostatic interaction), yellow (hydrogen bonding), green (metal coordination), and orange (cation–π interactions). Blue balls represent Fe3+ and Tb3+ ions. Download figure Download PowerPoint Moreover, the mechanical properties of ELP-K108-DSS fiber can be modulated by introducing Fe3+ ions. The uniaxial tensile tests showed the pronounced enhancement in mechanical properties compared with non-Fe3+ ion treatment (Figure 3g). Notably, the tensile strength and Young's modulus increased significantly to 159 ± 31 MPa and 17 ± 1 GPa, respectively ( Supporting Information Table S2), which is comparable with some natural spider silks.49 This behavior was attributed to the additional force of metal coordination chelation between DOPA ligand and metal ions (Figure 3h). Therefore, the results suggest that metal coordination chelation was crucial for the enhancement of the fiber's mechanical performance. Despite the similarity in water content in comparison with the fibers without metal coordination, the extensibility of fiber after metal treatment is smaller. This behavior can be ascribed to the formation of a coordination cross-linking network, limiting the movement of polypeptide chains, and resulting in the decreased extensibility. The introduction of metal-coordination bonds in the fibers facilitated the dehydration process and metal-coordinated cross-linking, resulting in high modulus but low extensibility. Therefore, the produced high modulus and low extensibility in the present system should be related to the formed internal network induced by the metal coordination. To understand the mechanical behaviors, surface morphology, and internal structure of the ELP-DSS fibers, SEM was performed. The SEM images of ELP fibers revealed distinctively different fibrillary structure features at different water content. For instance, the ELP-K108-DSS fiber showed smooth and uniform surface morphology and a deformed cross section at higher water content ( Supporting Information Figure S8a), whereas, a rougher surface was observed at lower water content or with metal ion treatment ( Supporting Information Figures S8b and S8c). Water molecules might act as plasticizers within the fibers thereby decreasing fiber roughness and imparting a smooth morphology to K108 fibers with high water content. These results indicate that the mechanical properties of ELP fibers can be manipulated by introducing metal coordination and varying water content. Other supramolecular interactions also play a crucial role in improving the mechanical performance of ELP fibers (Figure 3h). As discussed above, 1H NMR data ( Supporting Information Figure S2) showed that approximately 5% of lysine moieties is not complexed with the DSS surfactant molecules, indicating that the free, positively charged lysine residues are potentially improving fiber mechanics due to the cation–π interaction. When preparing an ELP-DSS complex in a molar ratio of lysine to surfactant of 1∶5, a stoichiometry of 4.4 DSS surfactant molecules per lysine of the ELP is verified by the 1H NMR spectrum ( Supporting Information Figure S9). However, this ELP-DSS complex will not spin into fibers. It is known that cation–π interaction plays a vital role in natural mussel plaque adhesives.42,50,51 Therefore, the cation–π interactions between unoccupied positively charged lysine and adjacent phenyl rings of DSS might be present in the ELP-DSS fiber. Moreover, complexing cationic ELP with a surfactant lacking the phenyl moiety (by using sodium dodecyl sulfate [SDS]) does not result in the formation of fibers. This indicates the importance of hydrogen bonding and cation–π stacking in the ELP-DSS fibers (Figure 3h). These results strongly suggest that in our supramolecular ELP-DSS fibers the long macromolecular backbone combined with the multiple supramolecular interactions, such as hydrogen bonding, electrostatic interaction, metal coordination chelation, and cation–π interaction, play a crucial role in improving the mechanical performance of ELP-DSS fibers. It is worth noting that the ELP-DSS fibers have additional unique properties. First, the ELP fibers exhibit high plasticity, forming helical structures by wrapping along a glass rod under ambient conditions (Figure 4a). This behavior is like a tendril host particular to helical structures that produces rapid and tunable mechanical actuation.52 The photograph of ELP-K72-DSS fiber revealed the R (right hand)/L (left hand) configuration, which was further confirmed by optical microscopy (Figures 4b and 4c). ELP-DSS fibers retained photoluminescence properties after Tb3+ ion treatment, further confirming metal chelation within the fibers. Interestingly, stretched ELP-K72-DSS-Tb complexes can be shaped into multiple patterns, including fibers, five-pointed stars, and "RUG" letters (Figures 4d and 4e). These patterns emitted green fluorescence when exposed to UV irradiation (λexc = 254 nm). Overall, the regulation of photoluminescence that our strategy offers a simple to biological fibers with multiple Figure | and fluorescence behaviors of the ELP-DSS fibers. (a) Photograph for the helical structure of the ELP-K72-DSS fiber which is also confirmed with optical analysis (c) A helical structure with was also formed in the ELP-K72-DSS fiber. (d and The ELP-K72-DSS-Tb fibers with different patterns and strong under UV irradiation (λexc = 254 nm). Download figure Download PowerPoint In this we have and and mechanically strong fibers by electrostatic complexation between positively charged ELP and charged DSS surfactant. The fibers exhibited tunable mechanical properties of interactions. Particularly, a Young's modulus of 17 GPa can be reached in fibers when treated with Fe3+ ions, which is comparable with some natural spider Moreover, ELP fibers exhibited significant plasticity, and photoluminescence properties. In to protein fibers, the mechanical performance of ELP fibers can be manipulated by multiple supramolecular interactions, including electrostatic interaction, metal coordination chelation, hydrogen bonding, and cation–π interactions. 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