Squid Sucker Ring Teeth - American Chemical Society

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Squid Sucker Ring Teeth: Multi-Scale Structure-Property Relationships, Sequencing, and Protein Engineering of a Thermoplastic Biopolymer Shuhui Hiew, and Ali Miserez ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00284 • Publication Date (Web): 08 Aug 2016 Downloaded from http://pubs.acs.org on August 21, 2016

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ACS Biomaterials Science & Engineering

Squid Sucker Ring Teeth: Multi-Scale Structure-Property Relationships, Sequencing, and Protein Engineering of a Thermoplastic Biopolymer Shu Hui Hiew1,2 and Ali Miserez 1,2,*

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School of Material Science and Engineering, Nanyang Technological University (NTU), 50 Nanyang Drive Singapore 639798. 2 Center for Biomimetic Sensor Science, NTU, Singapore * [email protected]

Abstract The arms and tentacles of Decapodiform cephalopods (squids and cuttlefish) are lined with suckers, each of which contains embedded Sucker Ring Teeth (SRT), which are used by the animal for prey capture and handling. SRT exhibit intriguing physico-chemical and thermo-mechanical characteristics that have so far not been observed in other protein-based biomaterials. Notably, despite their comparatively high mechanical properties, SRT are almost fully soluble in chaotropic solvents and can be readily reconstituted after solvent evaporation into three-dimensional structures. SRT also exhibit thermoplastic characteristics: they can be melted and re-shaped multiple times with no –or only minimal– loss of mechanical performance post-processing. Intrigued by these unusual material characteristics, in recent years we have conducted in-depth fundamental studies to unveil structure/property relationships of SRT from the molecular (genetic) level to the macroscopic scale. These investigations have demonstrated that SRT are entirely assembled from a protein family called “suckerins” that self-assemble into semicrystalline polymer infinite networks. Suckerins are block co-polymers at the molecular level, whose closest analogy appears to be silk fibroins, although significant differences exist between these two protein families. Parallel to these studies, there have been efforts to mimic and engineer suckerins by protein engineering and to demonstrate potential applications through proof-of-concept studies, with a focus on the biomedical field. Both fundamental aspects and emerging applications are presented in this short review. Given the rather unusual source of this model structure, we start by a brief historical account of SRT and suckerin discovery. Keywords Squid, Sucker Ring Teeth, Suckerin, β-sheets, Thermoplastic, Semi-crystalline, Block co-polymers, Protein engineering

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1. Historical perspective A common question from scientific (and non-scientific) colleagues when we present our studies on squid Sucker Ring Teeth (SRT) is “why did you even start studying this material in the first place“? Many people wonder how one would even know about the existence of such a biological material. This is certainly a legitimate question. While the study of biological materials has steadily increased in the past decade, with a growing number of scientists coming from the Physical and the Life Sciences (interestingly enough, the latter community appears less represented than the former in the field), one must acknowledge that conducting comprehensive research on what appears at first sight like an esoteric protrusion of cephalopods’ flexible appendages (arms and tentacles) may not be the most obvious research topic! In the search for model biological materials with high mechanically robustness, more obvious examples would come to mind, e.g. seashells, antlers, or silks1 to name just a few. Likewise, functional structures such as super-hydrophobic surfaces, for which more immediate engineering applications can be envisioned, are naturally drawing the attention of scientists. Interestingly, first descriptions of SRT from Humboldt (jumbo) squids date back to the 1850’s expedition2 by French naturalist Alcide D’Orbigny, who made beautiful drawings of SRT and other squid appendages, shown in Fig. 1. As often in curiosity-driven Science, research on SRT started from a serendipitous event. One of the co-authors (A.M., then a post-doctoral fellow at the University of California, Santa Barbara, UCSB) was investigating the intriguing mineral-free hard beak from Humboldt squids (Dosidicus gigas, D. gigas).3-5 A collaborator (James Weaver, currently at the Wyss Institute for Biologically Inspired Engineering at Harvard University) had collected SRT from a recent field trip and wanted to know how much chitin was present in these teeth, since invertebrate biology textbooks referred the teeth as “chitinous”. Upon acid hydrolysis, chitin decomposes into monomeric glucosamine units, which can be readily detected by Ninhydrin-based amino acid analysis.3 Surprisingly, no chitin was found in the teeth, and instead only standard amino acids were detected in the chromatograms. Since the amino acid composition indicated that SRT were mostly made of proteins, routine polyacrylamide gel electrophoresis was conducted. The analysis revealed additional unusual features appearing during sample preparation. The analysis revealed additional unusual features appearing during the sample preparation. Once the teeth were soaked in 5% acetic acid/6M urea (a common protein extraction solution for extra-cellular tissues), they almost fully dissolved. This is a very unusual behaviour, because sclerotized hard tissues, such as insect cuticles, hard jaws from marine worms6-8 or spider fangs,9 are extremely resistant to even the strongest denaturing cocktails. Usually, the extraction yield is just a few percents. Such intractability in fact remained a major reason why full-length sequences of most hard sclerotized hard tissues has eluded us for many years, although recent advances in Next Generation RNA-sequencing (RNA-seq) coupled with proteomics have largely lifted such constraints, as described in the next Section.10 In contrast, SRT were almost fully ACS Paragon Plus Environment

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soluble and resulted in sharp, distinct electrophoresis bands. That the sample was almost fully dissolvable in weak acids/urea mixtures suggested that inter-chain covalent cross-linking, the chemical strategy behind sclerotization, was absent and that, instead, the material was stabilized through the cooperative action of weak intermolecular interactions. Additional clues came from treatment in formic acid, a strong hydrogen bond breaker, or when heating the sample in boiling water, which resulted in SRT exhibiting a soft melt texture. However, SRT stiffness was regained upon cooling. A first series of ultrastructural and nano-mechanical studies were conducted by Miserez and Weaver and the findings were published in 200911. In the next step, we sought to identify and sequence SRT proteins, which was initiated in our newly established laboratory at Nanyang Technological University (NTU) in Singapore. The challenges mentioned above for full-length sequencing of extra-cellular biological materials were further exacerbated by the absence of both genomic and proteomic data on SRT (other than the overall amino acid composition). Using freshly collected samples, mRNAs from the sucker tissue surrounding the teeth were isolated by Paul Guerette, a senior scientist in our laboratory. Whereas the initial goal was to obtain the full-length sequences of the most abundant SRT proteins using traditional cDNA molecular cloning techniques, this strategy was quickly shifted towards RNA-seq upon realizing that this method is a powerful tool to assemble full-length transcripts de novo using massively parallel sequencing, which is particularly powerful for species without reference genomes. This work, which was conducted in close collaboration with Shawn Hoon from the Molecular Engineering Laboratory (MEL) at the Agency for Science, Technology, and Research (A*Star) in Singapore, led, within a few months to the sequencing of all SRT proteins (which were then re-named “suckerins”) not only from D. gigas, but also from other Decapodiform cephalopods. Paul Guerette’s expertise on spider silk molecular structure (he notably discovered that silk fibroins constituted a gene family12) proved critical because it greatly facilitated our task of identifying the modular peptide domains of the suckerins, leading to the important realization that suckerins share molecular homology with silks. Given how recently suckerins have been discovered in comparison to other well-known loadbearing proteins in the biomaterials field, such as silks,13 resilin,14 elastin,15 or mussel byssal threads,16 only a few studies and applications of suckerin-based materials have been reported thus far, which is the focus of this review. Only time will tell whether suckerins will gather a wider interest from the biomaterials community. From our biased perspective, we certainly believe that suckerins exhibit distinctive physico-chemical characteristics that we have only started to explore, and that there remains ample room for further developments. At a time where translational and immediate “impact” has become the central focus of many funding agencies and scientific fields, it is also our modest hope that this brief account will serve as a reminder that curiosity-driven science, with a keen eye for tangential explorations, remains an indispensable tool for unexpected discoveries. ACS Paragon Plus Environment

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2. SRT materials: mechanical properties and thermal processing In this short review, we describe the molecular and higher-scale assembly design of SRT and suckerins. Multi-scale structure/property relationships unveiled thus far are discussed, with an emphasis on the structural principles identified as responsible for SRT mechanical properties. As a flexible weapon, the squid’s arms come into contact with the prey and with a swift contraction of the sucker cups’ muscles, the cups latch onto the prey while the sharp SRT embedded within the sucker cups pierces the prey to anchor their grip.17 With the combination of hundreds of angled hooked teeth, prey items often suffer a futile struggling attempt to escape. The main biomechanical function of SRT is to firmly hold onto prey, which requires withstanding large dynamic compressive and shearing forces. Mechanical characterization by nanoindentation have revealed that D. gigas SRT exhibit a relatively high elastic modulus considering the absence of common strengthening phases, such as minerals,1, 18 metal ions,19-20 or even chitin.4, 21 In dry conditions, the elastic modulus ranges from 7.5 GPa to 4.5 GPa from the tooth periphery to the core, respectively.11 This modulus gradient is related to the presence of a nano-tubular gradient (Fig. 2A), with the tubular fraction concomitantly increasing from the periphery to the core (Fig. 2B). The porosity increase towards the tooth center can be correlated to relative changes in elastic modulus by considering the tubular architecture as a cellular material, for which power-law relationships have been developed for the modulus as a function of porosity content.22 Using these simple analytical models, it was found that the gradual change in modulus could be correlated to the measured tubular porosity. The use of cellular microstructures is a common strategy in various types of biological materials, such as cancellous bones, dentin, or horse hooves1 to name just a few illustrative examples. In these examples, the porosity is often used for weight minimization, with higher porosity content where mechanical stresses are less severe, namely in the bulk of the structure, whereas the outer surface usually remains dense. Given the relatively low porosity content in SRT and the fact that the protein weight density is also relatively small, it is unlikely that weight minimization is an important factor in the tubular design. This microstructure may be beneficial for crack deflection as a way to prevent catastrophic failure or perhaps the tubular structure is a by-product of SRT self-assembly. These are intriguing questions from a functional biology perspective, but they remain unanswered at the moment. Under hydrated conditions, these values drop between 2.75 GPa to 1.75 GPa, which is still considered quite high for a fully biopolymeric material in the hydrated state. These values, in combination with SRT hardness, are shown in Fig. 2C on a materials selection chart of biological materials.23 Compared to common engineering polymers,10 SRT under dry conditions appear stiffer than many synthetic polymers (such as polyamides (PA), polymethyl methacrylate (PMMA) and highmolecular weight polyethylene (PE)) as shown in Fig. 2D. Based on this combination of properties, the wear resistance of SRT has also been predicted to be quite high.


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Another distinctive feature of SRT is their thermoplastic behavior, which is very unusual for biological materials. This property was first suggested by realizing that SRT became pliable when heated in water, a characteristics that was reversible.11 This behavior was further demonstrated by heating crushed SRT in a small volume of water until the structure displayed a solid-liquid phase transition into a viscous melt that could be processed into complex shapes.10 Native SRT could be used directly for thermal processing into different 3D geometries, and this melting and shaping process could be repeated on the same sample without affecting the elastic modulus of the final material. A detailed understanding of the thermo-mechanical properties of SRT have been gained by correlating differential scanning calorimetry (DSC) with nanoindentation experiments, which demonstrated that the elastic modulus of SRT is highly dependent on the temperature as well as on the hydration state of the structure. Under dry conditions, the modulus remains high at 6 GPa and even increases up to nearly 12 GPa above 100°C because of water evaporation, while the modulus drops 50% to 3 GPa under hydrated conditions, and further decreases in the sub-GPa range as temperature increases.24 Further investigations of the elasticplastic response of reconstituted SRT as a function of water content25 have revealed a ductile-to-brittle transition as water is gradually removed from the network (Fig. 2E), which provides key insights into the role played by water as a plasticizer during thermo-processing, namely water makes SRT more flexible and less brittle. This distinctive combination of mechanical properties and thermoplastic behavior arises from the composition and molecular structure of the SRT, which is discussed below in further details. 3. SRT proteins: suckerins At the composition level, SRT are composed only of proteins, while chitin and minerals are notably absent from the teeth’s composition.11 Traces of sulfur and fluorine have been detected via EDS in the native material, though their role is unclear at this stage. Overall, the SRT proteins are biased towards 9 standard amino acids, the most abundant being Gly (37 mole %) followed by Tyr (14 mole %) and His (13 mole %), both of which can be exploited to either expand the properties of artificial SRT proteins or to modulate their solubility as a function of pH. Finally hydrophobic Leu, Ala, Val and Pro, as well as polar residues Thr and Ser, are also present in significant quantities. 3.1 Suckerin sequencing Suckerins have been sequenced using RNA-seq analysis26-28 in conjunction with proteomic techniques, including liquid chromatography tandem mass spectroscopy (LC-MS/MS) and traditional Nterminal Edman sequencing.10 In short, the translated assembled transcriptome from the suckers was used as an internal database that can be uploaded within the high-throughput mass spectrometry software PEAKS.29 In parallel, extracted suckerins from SRT were purified, digested, and subjected to LCMS/MS, to generate de novo internal sequences using PEAKS. By screening the de novo peptides against the transcriptome libraries, suckerin peptides can be identified with a high coverage against the


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translated sequences. RACE-PCR30 was eventually employed to confirm the full-length sequences. Other traditional proteomic techniques were also employed. For example, SDS-PAGE protein bands of SRT extracts were subjected to Edman sequencing, and the obtained N-termini were probed against transcriptome libraries, resulting in very quick suckerin identification from the transcripts. With the amino acid composition of the individual suckerins known (either in the purified form or from individual SDS-PAGE bands), their amino acid composition could also be probed against the libraries, and that resulted in quick sequence identification. Using this approach, a total of 21 suckerins were sequenced from the Humboldt squid31 with molecular weights ranging from ~5 to 57 kDa. In a comparative study, suckerins from distantly related Decapodiform cephalopods were also sequenced using the same techniques. 8 suckerin sequences were obtained from the Bigfin reef squid (Sepioteuthis lessoniana, S. lessoniana), while 9 were obtained from the Golden cuttlefish (Sepia esculenta, S. esculenta) (Fig. 3A and E). Humboldt squid SRT were thus found to contain a larger repertoire of proteins than the two other cephalopods, and almost all suckerins exhibited similarity in amino acid composition. Replicating our approach, others have sequenced suckerins from the common squid (Loligo vulgaris), although not all full-length sequences were obtained.32 3.2 Molecular design of suckerins A key feature emerging from this comprehensive sequencing study is that suckerins have a highly modular design that exhibits resemblance to silk proteins. Indeed, ampullate spider silk proteins are generally constructed from highly modular Gly repeats with alternating poly-Ala motifs.12, 33-34 Most suckerins are also composed of silk-like GGY repeats with alternating Ala-rich modules. More specifically, Humboldt squid suckerins are composed of consecutive repetitive modules [M1] and [M2], which can be classified as such: [M1] modules are rich in Ala, Val, His, Thr, and Ser residues, ranging from 10–15 residues in length, with the archetypal presence of Pro residues flanking these modules. The second iconic modules are [M2] modules, which are rich in Gly, Tyr, and Leu, and range from 20 to 40 residues in length.31 With a few exceptions, these two types of modules are ubiquitous in suckerins, and each module confers to the suckerin distinct characteristics. A significant amount of the repeats constituting the suckerins are hydrophobic in nature (enriched in Ala, Val, Pro, Gly and Leu residues). The other abundant residues are Thr, Ser, as well as the aromatic Tyr (14 mole %) and the slightly basic His (13 mole %), which are useful for chemical functionalization and processability, respectively. Owing to the versatility of the imidazole moiety, His residues play an important role in structural proteins by forming metal-coordination interactions19-20, 35 or covalent bonds with other residues4-5 to stablilize the network. However, these interactions have not been detected in SRT. The [M1] modules enriched in hydrophobic Ala and Val residues resemble the β-sheet forming Alarich repeats of silk proteins33 and are reminiscent of the peptide sequence GGVVIA, which contributes to


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cross-β structures in beta amyloids.36-37 On the other hand, [M2] modules rich in Gly, Tyr, and Leu residues, is reminiscent of the amorphous-forming sequences in dragline silk proteins.38 Pro residues have been observed to consistently flank the [M1] modules, acting as a divider between the [M1] and [M2] modules. The repeating unit of Pro-flanking [M1] modules followed by [M2] modules is reiterated through the full length of the suckerins (Fig. 3C and D), and observed across most of the highly conserved suckerins from different cephalopod species. For example in the case of D. gigas suckerin-19, Pro-[M1]Pro-[M2] repeats 11 times in the full sequence. Similar to silk proteins, the Ala-rich [M1] modules have been shown to form β-sheets, while the Gly-rich [M2] modules are thought to be mostly amorphous, such that the overall structure is similar to semi-crystalline synthetic polymers. Pro residues, which are known to be β -sheet disruptors,39-40 are strategically placed to restrict the dimensions of the β -sheets formed by [M1], resulting in β -sheets31 that are typically 10-11 amino acid long or 3-3.5 nm long. These nanoconfined β -sheet domains are embedded within the longer [M2] amorphous domains that provide flexibility and elasticity. Hence, with this semi-crystalline molecular design arising from the repeat peptide building blocks, a suitable combination of rigidity and elasticity is obtained within the SRT supramolecular network. While primary sequences were not analysed in detail for L. vulgaris31, a quick perusal of the reported sequences also confirmed the block co-polymer-like structure identified in our comprehensive studies. To date, the majority of biophysical data on suckerins have been obtained from recombinant D. gigas suckerin-19. There are three main reasons for this choice. First suckerin-19 is the most abundant protein in the D. gigas SRT. Second, its molecular weight (MW) of 39 kDa is mid-range between the smallest (~ 10 kDa) and the largest (~57 kDa) suckerins. And third, it exhibits the classical Pro-[M1]-Pro[M2] modular architecture, such that it represents an illustrative example of the suckerin protein family. However it should be emphasized that there are subtle variations within the suckerin gene family in terms of the length of modules [M] or the isoelectric points due to the occurrence of a few basic residues in some suckerins. The existence of this large number and size range of suckerins is postulated to help keep the protein complex soluble prior to assembly, and to help with packing during assembly. The roles of such variations, however, have not yet been explored and remain to be confirmed. 3.3. Self-assembly of suckerin peptide units By dissecting suckerin-19 down to its modular peptide building blocks, the role of each module and their inter-modular interactions can be individually assessed. Abundant repetitive short Ala-rich peptides and His-rich peptides from [M1] modules, as well as a few Gly-rich peptides from [M2] modules have been identified and studied in greater detail.


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Using a designed peptide interaction assay that applies combinatorial chemistry concepts,41-43 Alarich and His-rich peptides were identified to express significant binding interactions in both homo- and hetero- suckerin peptide interactions.44 These peptides exhibit a high tendency to self-assemble into larger secondary structures from solution as shown by DLS measurements over a period of several days. Further examination of Ala- and His-rich peptides via circular dichroism spectroscopy found that they both form polyproline II (PPII) and β -sheet secondary structures in solution, and FTIR spectroscopic studies revealed high β -sheet content both in solution and dried conditions. The major difference between the two types of peptides is their solubility. His-rich peptides tend to stay soluble over long incubation periods while Ala-rich peptides tend to aggregate easily. The micron-scale aggregates formed by Ala-rich peptides in solution are fibrillar as revealed by electron microscopy and they demonstrate elevated stability against sonication and strong denaturing agents such as 8M urea. Uniform and high amide backbone proton protection factors of each residue in the Ala-rich peptide fibers were observed from 1D hydrogen-deuterium exchange NMR spectroscopy studies, indicating generally uniform participation in β -sheet hydrogen bonding from all residues in the sequence. With a high tendency to self-assemble into larger fibrillar structures, Ala-rich modules alone face a critical problem of premature aggregation before being processed into larger functional structures. This behavior of the Ala-rich modules is brought about by their hydrophobicity and amyloid-like ability to form β-sheet seeds, which propagate to form larger β-sheet-rich networks. However with the flanking of His-rich modules, the mildly basic His residues (which are protonated under acidic conditions) may help to maintain the proteins in solution before secretion, possibly preventing early aggregation of the Alarich domains, in turn allowing the self-assembly of β -sheet-rich fibrils from solution. The interaction assay also indicated that self-assembly of His-rich suckerin peptides is the strongest close to neutral pH, whereas fewer interactions were observed under acidic conditions. Therefore the current hypothesis for self-assembly of suckerin into the final SRT structure is that upon secretion in the external environment, de-protonation of His residues occurs, eliminating charge-charge repulsion and driving β -sheet formation. As for Gly-rich peptides, they generally exhibited low to moderate homo- and hetero- peptide interactions. In full-length suckerins, a classical Gly-rich [M2] module typically contains more than 50% Gly residues. Since Gly only has a single hydrogen in its side chains, it is the least sterically hindered amino acid, thus conferring significant flexibility to [M2] modules.

3.4. Homology and conservation of suckerins In a comparative study across three species of cephalopods shown in Fig. 3A (D. gigas, S. lessoniana and S. esculenta),31 suckerins were identified to be encoded by members of a multigene family. Typical signal peptide lengths of suckerins are ca. 17 – 23 residues, while amino acid compositions appear very ACS Paragon Plus Environment

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similar (Section 3.2), with sequence homology between the suckerins exhibiting ~20 – 90% identity. Large scale modular architecture of Pro-[M1]-Pro-[M2] units (Fig. 3C and D) were also observed in the majority of suckerins, with variation in modular design for others. The frequency and length of [M2] Glyrich modules containing the smaller peptide building blocks GGY and GGLY also vary between the different suckerins, which could result in differences in mechanical behavior and self-assembly of SRT. Similar to studies carried out on different species of spiders,45-46 the suckerins’ modular design from all three species could be used to construct phylogenetic trees, which demonstrated that suckerin genes can be classified into 6 different clades (Fig. 3E), and that the origin of suckerins traces back to the Devonian period. Genetic divergence of the suckerin proteins of these three species includes gene duplication and segmental expansion or deletions. This can be observed clearly in D. gigas via analysis of the strikingly similar exon-intron patterns between suckerins of different lengths and modular architectures, suggesting that suckerin genes were derived from a common ancestral gene. Future transcriptomic and genetic analyses of suckerins from a wider range of cephalopods should provide interesting insights into molecular evolutionary processes behind SRT functional mechanics. For instance, it is intriguing that Humboldt squids have evolved a larger repertoire of suckerins (21 of them) than the bigfin reef squid (8 suckerins) and the golden cuttlefish (9 suckerins). Such variations could be related to their aggressive predatory strategies, by allowing Humboldt squids to construct stiffer and stronger SRT for prey capture using a higher number of suckerins, but this hypothesis remains to be verified.

4. Interplays between the molecular design and nano-scale structure 4.1 Molecular scale: WAXS, FTIR, and Raman spectroscopy Following the initial investigations of SRT from Humboldt squid,11 subsequent studies have revealed deeper insights into their biophysical and hierarchical structural characteristics. As a biomaterial constructed exclusively from proteins and apparently assembled together via weak hydrogen bonding, the intriguing question is: what makes the structure robust against high compressive and shearing forces? The answer lies with the molecular structure and supramolecular packing of suckerins, and is directly related to their modular organization discussed above. Raman spectroscopy on embedded and microtomed cross-sections of native SRT revealed the extensive presence of β-sheets throughout the teeth, which is notable by the location of the Amide I band centered at a Raman shift value of 1666 cm1 10

. Silks are well-established to be reinforced by β -sheet crystals.47-50 But in contrast to silk, where β -

sheets are highly aligned along the fiber axis,51-53 β-sheets in SRT are randomly oriented, as revealed by polarized Raman spectroscopy (Fig. 4A). This prevalence of β -sheets was confirmed by ATR-FTIR studies and even more clearly by Wide-Angle X-ray Scattering (WAXS) investigations, which further corroborated the isotropic orientation of β -sheets. WAXS revealed a circular scattering pattern of equal intensities at ACS Paragon Plus Environment

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all azimuths (Fig. 4C), indicating random orientation of crystalline domains within the native SRT.24, 31 Integration of all azimuthal53-54 angles gave reflection positions similar to that of silk, with the most intense peaks coming from the combination of [120] and [200] reflections, as well as the presence of [100] and [002] reflections. Comprehensive molecular modeling studies by Buehler and co-workers47, 49 have revealed that βsheets of restricted nano-scale dimensions play a fundamental role in enhancing the materials’ mechanical robustness, including strength and fracture toughness at the molecular level. In silk, the highest energy barrier that could be achieved in a uniformly loaded H-bond assembly, such as that between two β -strands, is restricted by an intrinsic upper limit of 3 – 4 H-bonds, whereby the highest shear resistance was computed over a length of 3.1 residues.47 These 3 – 4 H-bonds under homogenous shear loading can break concurrently, with a uniform deformation of multiple bonds contributing to the energy barrier. Exceeding the critical geometric strand length, the shear strength drops rapidly and the system is prone to localization of deformation and rupture of H-bond clusters. Thus, computational modeling suggests that there exists a critical size providing optimized mechanical performance, with estimated dimensions of 1 – 2 nm (along the peptide backbone) by 2 – 4 nm (in the H-bond direction).55 Analysis of the WAXS patterns in suckerins using the Debye-Scherrer equation estimated that β-sheets exhibit dimensions of 2.4 – 2.6 nm in the H-bond direction and 3 – 3.5 nm along the peptide backbone.31 An amorphous halo was also detected, implying the presence of a semi-crystalline structure at the molecular scale. β -sheets in SRT are thus predicted to be ~8-10 amino acids long and 5 strands wide, existing as uniform and size-restricted nanocrystals, that are randomly oriented within an amorphous matrix. The β -sheets size correlates well with the mean number (10-11) of residues flanked by β -sheet breaker Pro in the [M1] modules, which led us to suggest that β-strands are mostly made from the [M1] modules (disregarding residues directly adjacent to Pro due to their likelihood to be involved in disordered or β -turn structures), whereas the longer [M2] modules constitute the amorphous phase. The isotropic orientation is fully consistent with the biological function of SRT: as opposed to spider dragline silks38 whereby the aligned β -sheet crystals must sustain uniaxial tension, SRT are subjected to a combination of compressive and shearing stresses in vivo. Thus an isotropic orientation of load-bearing molecular blocks ensures that the structure can sustain stresses in multiple directions, with β -sheets mostly providing rigidity and the amorphous domains ensuring an adequate degree of flexibility.

4.2. Nano-scale: SAXS In contrast to the molecular level, small-angle x-ray scattering (SAXS) studies on native SRT have revealed an anisotropic organization at the nanoscale (Fig. 4B). 2D SAXS patterns were obtained for


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entire teeth at ca. 60 µm spatial resolution and exhibited an ellipsoidal shape with enhanced scattering intensity along the equatorial direction, denoting a preferred orientation of fibrillar elements that generally follows the contour of the teeth.24 Radial integration of the 2D SAXS patterns led to the distinction of several orders of Bragg reflections that were generally consistent with a hexagonal packing of elongated nanofibrils (Fig. 4D). An average inter-fibrillar distance of ca. 12.5 nm was calculated in this hexagonal packing. The multi-scale structural model of SRT, emerging from the combined molecular and nano-scale studies, is that of nanofibrils formed by the suckerin proteins. These fibrils are made up of randomly oriented 3 to 5 nm β -sheet crystals embedded within an amorphous protein matrix, with the nanofibril units packing into a hexagonal lattice in the native state (Fig. 4D). In the living animal, SRT are likely to be loaded in a bending regime when catching prey. Therefore our current hypothesis for the functional role of the nanofibrillar architecture –with nanofibrils oriented parallel to the teeth contour– is that they provide enhanced bending rigidity. A bending moment also implies that some regions of the SRT are loaded in compression, and that there are also significant shearing stresses in the structure. Having isotropically-oriented β -sheets as building blocks of the nanofibrils may ensure that they can sustain compressive loads, thereby preventing premature nanofibrils buckling 4.3 Thermo-mechanical properties and processing Thermal gravimetric analysis (TGA) indicates that SRT contains about 13 wt.% water, and that the teeth are stable up to 250°C. At the molecular scale, thermal transitions were probed by WAXS, as illustrated in Fig. 4D. Slight disordering along the β -strand direction was noticed due to water content removal around 80°C, while melting of the nanoconfined β -sheets was observed from the broadening of the main β -sheet reflection at 230°C.24 This reflection further decreased and disappeared as the temperature was raised to 250°C - 300°C, indicating polymer network degradation above 250°C. At the nanoscale, SAXS profiles indicated loss and re-organization of the ordered hexagonal packing at 80°C, which has been attributed to water removal from the network. As the temperature increased, new reflections appeared, suggesting the rearrangement and compaction of the nanofibrillar lattice. Finally the reflections disappeared completely above 230°C, indicating structural collapse. After thermal processing, it is interesting to note that the isotropic WAXS signal remained almost unchanged, whereas the higher order reflections of the SAXS pattern disappeared. These features provide structural explanations for the thermo-plastic behavior of SRT: since thermal treatment leads to loss of nano-scale packing while the molecular scale remains intact, the emerging model is that the amorphous regions locally melt during thermal processing, while the β -sheets domains remain rigid, leading to collapse of the nanofibrillar network (Fig. 4D). Upon cooling, the nanofibrils are not reformed, but the material largely maintains its mechanical properties25 because the load-bearing β -sheets are not affected by heattreatment. This model also implies that it should be possible to process elongated fibers, by taking


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advantage of the viscous nature of the (collapsed) amorphous domains which could be elongated during extrusion (Fig. 4FE). This was indeed confirmed in proof-of-concept experiments where 1.5 mm and 300 μm diameter SRT filaments were formed through vacuum casting and thermal extrusion-based 3D printing (Fig. 5).24 Optimization of processing conditions can be obtained using a plasticizing agent that facilitates chain movement of amorphous domains at the molecular and the nano scales. 5. Emerging applications of native SRT and recombinant suckerins Concurrently to the establishment of multi-scale structural/property relationships of SRT, materials made of suckerins have been prepared using both native and recombinant suckerins. These proof-ofconcept developments have so far mostly been conducted with a focus towards biomedical applications. 5.1 Recombinant production and purification With suckerin sequences elucidated, the natural next steps have been to express and purify the proteins, investigate their physico-chemical properties, and exploit these characteristics to develop novel suckerin-based materials. Most of the work thus far has been conducted using D. gigas suckerin-19, because as mentioned above, it is representative of the block co-polymer structure of the suckerin protein family. Given the molecular similarity to silks, much of the work using recombinant suckerins has been inspired by previous research on recombinant silk fibroins. An increasing number of biomedical and engineering applications have been described for artificial silk and other structural recombinant proteins, and their eventual commercial success will likely depend on improving protein production yields and reducing costs. This optimization remains somehow empirical because it is challenging to predict which cell lines and conditions will lead to high yields. In the case of silk, for instance, the highly repetitive GCrich gene sequences can be a detrimental problem during the production of these silk proteins, hence limiting the efficiency of production in E. coli.56-58 Another issue arises from the large molecular sizes of the silk proteins, for example the B. mori silk heavy-chain fibroin protein is approximately 350 kDa while the light-chain is ca. 26 kDa, and these proteins have the tendency to form insoluble aggregates due to their premature assembly into β -sheet structures.59-60 Nevertheless some of these challenges have been addressed in recent years and several groups are now producing silk fibroins in reasonably high yields,58,61 including high MW silk. For example, Xiao et al.62 have employed a metabolically engineered expression host in order to obtain silk fibroins with MW as high as 285 kDa, which resulted in elevated mechanical properties compared to lower MW fibroins. In other studies, fibers from recombinant spider silk achieving the same mechanical toughness as the native silk have been obtained,63 whereas a wide range of functional materials (films, hydrogels, sponges, adhesives) with tailorable properties have been reported from silk fibroins obtained from the milk of transgenic goats.64


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In the case of suckerins, the MWs of suckerins from Humboldt squid, bigfin reef squid, and cuttlefish range from 4kDa to less than 60 kDa, almost all of which exhibit the bi-modular, block co-polymer-like structure.31 This provides a large palette of suckerins to choose for recombinant expression and only a handful has so far been explored; however the challenges mentioned above such as the genetic instability or protein aggregation post-purification are largely avoided in suckerins. Given the large number of suckerins identified and their significant differences at the primary structure level, it is anticipated that the range of physico-chemical characteristics and applications could be expanded further. In our initial studies, both poly-His tagged suckerin-19 and full-length suckerin-19 were expressed in E. coli BL21 (DE3) cells and Rosetta cells.65 Successfully expressed His-tagged suckerin-19 was purified via immobilized metal affinity chromatography (IMAC) followed by TEV protease cleavage at the designed cleavage site, and a final ion exchange fast protein liquid chromatography (FPLC) step. However, this method proved too time-consuming and alternative approaches have been developed. Instead of the tedious chromatography processes, one can take advantage of the slightly basic isoelectric point (IEP) to conduct IEP precipitation by simple dialysis against a buffer close to the protein’s IEP. Full-length suckerin-19 could be obtained with high purity when salted-out with a pH 8.4 buffer containing denaturing agents, providing an alternative method that required fewer steps and reagents. Another technical improvement to enhance protein yield is to use microfluidization for cell lysis, which has so far led to expression yield of purified proteins in the range of ~50 mg/L in flask culture. While such yield is sufficient at the laboratory scale, it is clear that further improvements will be necessary in the future. However, till date only expression in E. coli has been attempted and no specific efforts to optimize the process have been undertaken, leaving ample room for optimization (fermentation conditions, metabolic engineering, etc). Likewise bioprocessing methods using insect or yeast cells have yet to be explored and are anticipated to further enhance production yields. Based on work conducted with more established proteins, it is not unreasonable to expect yields in the range of 1g/L of cell culture in the near future. 5.2 Thermoplastic forming Owing to their thermoplastic characteristics, native SRT can be thermally processed and shaped into 3D geometrical structures. Under hydrated conditions, the glass transition temperature (Tg) is in the range 30-40°C as observed by DSC measurements,24, 66 but is much higher in dry conditions. Theoretically, it is possible for Tg to be above the degradation temperature, meaning that thermo-processing is physically not possible in the absence of water or of another plasticizing agent. Water thus plays a key plasticizing role during thermo-processing by lowering the operation temperature at which SRT can be thermo-formed. Using these conditions, SRT have been casted into films, bulk samples25 or nano-pillars10. Plasticizing agents with a high boiling temperature, such as glycerol can also be added to the system to improve the mouldability of the melt and this approach was for instance applied to extrude SRT melt into ACS Paragon Plus Environment

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feed filaments with geometrical characteristics suitable for 3D printing applications (Fig. 5).24 A key aspect of this process is to maintain the processing temperature below the melting temperature of the

β-sheets, typically in the range 100-150°C. Critically, molded and re-constituted SRT could be recycled and re-processed many times without affecting the mechanical properties of the material.24-25 Exploiting these thermoplastic characteristics, SRT have also been processed into pressure-sensitive adhesives66, by processing the materials above Tg, which may find applications as bio-adhesives.

5.3 From soft gels to stiff films using Di-Tyr cross-linking The solubility of suckerin-19 is below 10 mg/ml in water, but it can be increased as high as 71 mg/ml in 5% acetic acid solution,65 with the solution forming a colloidal suspension of nano-scale particles that remain stable at 4°C over a two weeks period. DLS analysis has indicated nearly constant size distributions over this time period, and high zeta potentials of the colloidal solutions were measured (in general >30 mV), indicating high colloidal stability. Furthermore, suitable adjustments of the buffer conditions allow for the preparation of suckerin-19 nanoparticles with well-controlled sizes. This stability of suckerin-19 colloidal suspensions combined with their high solubility in aqueous-based solvents makes them favorable for further processing into various functional and structural materials. Exploiting the abundant Tyr residues located in the amorphous [M2] modules, di-Tyr cross-linking can be achieved using a photo-cross-linking method,67 and the cross-linking density can be varied to tune the final mechanical response. Using this approach, di-Tyr cross-linked gels (Fig. 6A) and films exhibiting a wide range of elastic properties (Fig. 6D) could be prepared, from soft gels with storage modulus in the subKPa range to stiff films with elastic modulus above 1 GPa under hydrated conditions.68 The implication is that the suckerin-19 could, in principle, match the elasticity of a wide range of tissues, which could be exploited in biomedical applications and tissue engineering. For such applications, the assessment of biocompatibility is essential. Suckerin-19 films have been evaluated for biocompatibility and such assays have so far indicated negligible in vitro cyto-toxicity against various cell lines, including human dermal fibroblast cells (HDF) and mesenchymal stem cells (hMSCs), even leading to faster cell proliferation than on commercial tissue culture plates. However biocompatibility studies have so far remained limited and comprehensive in vivo studies are needed in order to obtain a more thorough assessment of the suitability of suckerins for biomedical applications. From a polymer physics perspective, it is of interest to note that suckerin-based materials exhibit several features that can be attributed to their modular architecture. For example, the rheological behavior of suckerin-19 gels were consistent with a semi-flexible polymer network in which β -sheets form the rigid elements and the amorphous domains the entropic springs. For suckerin-19 films, the relationship between the modulus and the cross-link density is peculiar, since higher cross-linking density led to films with a lower modulus (Fig. 6B and C).68 This “non-classical” dependence of the modulus on


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the cross-link density was reconciled by observing that higher cross-linking density resulted in a lower β sheet content (Fig. 6C), and thus to lower moduli since β -sheets are the dominant load-bearing building blocks in the suckerins. This phenomenon was explained by the fact that Tyr residues, which are the cross-linking sites, are strictly located in the amorphous domains. When the cross-linking density increases, the backbone flexibility of the amorphous domains becomes restricted, thus limiting the formation of adjacent [M1] modules into β -sheets (Fig. 6C). In other words, β -sheet formation is frustrated, resulting in films with a lower modulus at higher cross-linking density. It would be of interest to test whether this molecular design could be applied to other semi-crystalline structures made of rigid supramolecular blocks.69

5.4. Suckerins as substrates for the growth of gold nanoparticles Owing to their intrinsic high content of Tyr residues (up to 15 mole %), suckerins (suckerin-12 and suckerin-19) can be used as macromolecular templates to trigger the growth of metallic nanoparticles exhibiting the classical plasmonic effect,70 with the Tyr side-chain playing the role of reducing agent when the pH of the solution is raised above its pKa. Growing Au nanoparticles with controlled sizes using proteins has previously been demonstrated, for instance with BSA71 as well as silk.72 In the latter case, Tyr residues were identified as the moieties promoting the reduction of HAuCl4 salts into Au nanoparticle suspensions, which remained highly stable owing to silk fibroins surrounding the nanoparticles and preventing aggregation by steric repulsion. The same mechanism is responsible for inducing the formation Au nanoparticles from suckerin solutions.73 In addition, changing the suckerin concentration allowed the preparation of Au nanoplatelets of triangular or hexagonal shape (Fig. 7A and B), which is especially useful for surface-enhanced Raman spectroscopy analysis.74 The formation of Au nanoplatelets from biomacromolecules is usually triggered by acidic residues that preferentially adsorb on {111} facets of Au crystals,71, 75 therefore suppressing growth in the direction and promoting planar geometry. Since there are no acidic residues in suckerin-12 and suckerin-19 employed in this study, the planar geometry must be attributed to a different mechanism. One possibility is that Tyr residues that are weakly protonated at the pH above their pKa trigger the reduction of Au salts, such that charged Tyrosyl may adsorb onto {111} facets in the same fashion as Asp or Glu residues at lower pH. The other plausible explanation is linked to the high β -sheet content, especially at high concentration of suckerins,65 where

β-sheets may also exhibit enhanced adsorption onto {111} facets, a mechanism supported by molecular dynamic simulations which showed that β−sheets exhibit a strong affinity for {111} planes.76 The ability to grow Au nano-objects from suckerins opens several opportunities in the field of nanotechnology. For example, it allows for the immobilization of Au nanoparticles onto solid substrates without aggregation, which often causes the immobilized nanoparticles to lose their size-dependent ACS Paragon Plus Environment

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optical and electronic properties.77 Another noteworthy feature demonstrated in Ref. [73] is the capacity to cast high concentration of suckerin solutions into micro- or nano-templates followed by solvent evaporation, leading to the formation of nano-structured suckerins templates (Figs. 7C and D). In the subsequent step, a thin Au film can be grown over the suckerin template by simple dipping into an Au salt solution, with the suckerin coating playing the role of a redox active solid substrate (Figs. 7E and F). Suckerins can therefore serve as a potential alternative to evaporation and sputtering techniques for the selective spatial deposition of Au films onto solid surfaces. A further expansion of this application would be to use suckerins as sacrificial templates that could be readily solubilized following Au nanoparticle film growth, leaving only the metal film behind. This may be employed in micro- or nano-fabrication or to prepare gold aerogels, similar to what has been achieved using amyloid templates.78

5.5. Short peptides of suckerin: amyloid-like structure and potential applications Biophysical measurements of short suckerin peptides44 point towards the formation of cross β structures, the classical feature of amyloids fibrils.79-81 Self-assembly studies of the short suckerin peptides showed that they quickly assemble into high aspect ratio fibers. Thus, suckerins provide a wide range of short peptides that can be used to create functional amyloid-based materials, for which there have been numerous developments in recent years.82 For example, recent applications of functional amyloids include hybrid membranes for water filtration purposes, wherein protein amyloid fibrils in combination with activated porous carbon can effectively remove pollutants such as heavy metal ions and radioactive waste from water at low cost.83 Other interesting applications include multi-functional hybrid nanocomposites of amyloid fibrils and gold platelets crystals. These hybrid structures have watersensing properties and possess surface plasmon resonance features, potentially making them useful as pH sensors.84 As smaller amyloid-like building blocks, Ala-rich peptides from suckerins spontaneously assemble to form short amyloidogenic fibers, offering the possibility to develop amyloid-based functional materials and devices as described above. Longer Ala- and His-rich suckerin peptides have also been prepared into micro-fibrils using mild processing conditions, and exhibited similar robust mechanical properties as the native SRT. These fibers are mechanically tough and may represent promising candidates as load-bearing building blocks for nano-composites. Certain Gly-rich suckerin peptides also have the capability to self-assemble into hydrogels (Fig. 8) in aqueous solutions (unpublished data). Since the length of the peptides is short, synthesis and purification processes are simple and high yields can be obtained efficiently, which is favorable for mass scale production. With the easy processing of the peptide into hydrogels and the advantage of being a biodegradable material, the usage of these materials in the biomedical (for instance as 3D cell culture substrates) or cosmetic fields are envisioned.


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6. Conclusion Since our initial report describing the biochemistry and mechanical properties of Humboldt squid 11

SRT, we have broadened our fundamental understanding of this protein-only extra-cellular tissue and gained deeper insights into the structural principles responsible for its intriguing properties, from the meso-scale all the way down to the genetic level. This understanding has provided valuable lessons with regard to how Nature employs relatively weak building blocks to construct robust materials using supramolecular and multi-scale assembly design principles. A central feature of native SRT is the absence of inter-chain covalent crosslinks, which enables SRT to be dissolved in weak acidic or alkaline buffers, making SRT and suckerins an original renewable and biodegradable natural biopolymer resource. Using these insights, recombinant suckerins have been engineered into various functional and structural materials, each taking advantage of suckerins’ molecular design. Given the versatility of SRT and its suckerin proteins, a wide range of further applications may be developed in the near future, although only a fraction has been explored thus far. Ranging from the biomedical field to biophotonics to additive manufacturing, suckerin proteins and peptides may be incorporated into composite materials or nano-scale devices, or engineered into functional or structural materials. Protein-based materials usually display high biocompatibility with low cytotoxicity, and are biodegradable with natural amino acids generated as metabolic products.85 Precise control of the protein sequence down to the single amino acid level can be achieved and bioactive domains can be incorporated, such as cell-recognition or enzymatic degradation site domains.86-87 Chemical modifications of side chain functional groups is also readily achievable,88 therefore making proteins versatile candidates in the biomedical field. Recombinant suckerins have so far shown excellent biocompatibility and no cytotoxicity, and the next step will be to expand such studies in vivo, with the goal to further explore their applicability in the bioengineering and biotechnology fields. Potential uses include tissue engineering scaffolds and drug delivery vehicles, biosensors and molecular switches, printed wearable electronics, or bioelastomer adhesives for wound healing or surgical sutures. We have only begun to investigate these opportunities and we hope that this mini-review will stimulate others to explore the specific physico-chemical and thermo-mechanical properties of suckerins. With nearly 50 suckerins discovered thus far –and likely many more yet to be sequenced– combined with their ease of recombinant expression, we have a broad molecular biotoolbox at our disposal to expand the above-mentioned possibilities. Author information Corresponding Author: *Email: [email protected] Acknowledgements We gratefully acknowledge financial support from the Singapore National Research Foundation (NRF) through a NRF Fellowship awarded to A.M., and from a Singapore Ministry of Education (MOE) ACS Paragon Plus Environment

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Academic Tier 2 grant (Grant No. MOE 2011- T2-2-044). We thank our collaborators and colleagues that have contributed to the work described in this review over the years, in particular James Weaver (Wyss Institute, Harvard University), Paul Guerette (formerly at NTU, now at BoltThreads), Shawn Hoon (Molecular Engineering Lab, MEL, A*Star), Admir Masic (previously at the Max-Planck Institute for Colloids and Interfaces, now at MIT), Byrappa Venkatesh (A*Star), Bram Cantaert (formerly at NTU, now at the University of Tokyo), Dawei Ding (NTU), Akshita Kumar (NTU), and Brendan Orner (previously at NTU, now at King’s College London). We also thank Ho Chin Guan and Tan Bee Yi (NTU) for their contribution to the suckerin peptide hydrogel work. We would like to thank Brian J. Skerry for granting us permission to use his work (photo of a pair of Humboldt Squids) in our TOC.


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Figure Legends

Figure 1. Drawing of Humboldt squid (Dosidicus gigas) and its “hard tissues” appendages by French naturalist, Alcide D’Orbigny. (Voyage dans l’Amerique Meridionale: Tome Neuvieme. 1847.)

Figure 2. Mechanical properties and structure of Humboldt squid sucker ring teeth. Scanning electron microscope (SEM) image showing (A) the nano-tubular structure of a SRT fracture section and (B) the porous tubular cross-section of the teeth with inset presenting a scanning probe microscope image of the SRT’s porous cross-section using a nanoindentor in the scanning mode. (C) Materials selection chart (Ashby plot of biological materials with SRT core and periphery indicated, and. (D) Ashby plot of engineering materials such as polymers, metals and ceramic materials, with dried native squid sucker ring teeth (values ranging from core to periphery) indicated. (E) Tensile test curves performed on melt-cast processed SRT ribbons at different humidities, revealing ductile-to-brittle transition from wet to dry samples. Panel C reproduced from Ref. [23] with authorization from Elsevier (Copyright 2013 Elsevier). Panel D reproduced from Ref. [10] with authorization from Nature Publishing Group (Copyright 2013 Nature Publishing Group). Panel E reproduced from Ref. [25].

Figure 3. SRT and their constitutive suckerins from various cephalopod species. (A) Scalar comparison of three cephalopod species, Humboldt squid (D. gigas)- top, Bigfin reef squid (S. lessoniana)- middle and Golden cuttlefish (S. esulenta)- bottom, and their respective SRT (B). (C) Modular architecture of a representative Humboldt squid suckerin (suckerin-12) showing regular Pro placement, flanking [M1] modules. (D) Distribution of the number of amino acid residues between consecutive Pro for all Humboldt squid suckerins. (E) Phylogenetic tree depicting relationships of all known suckerin proteins, with the classification of six clades. All panels reproduced from Ref. [31] with authorization from American Chemical Society (Copyright 2014 American Chemical Society).

Figure 4. Molecular design and nano-scale structure of SRT. (A) Raman spectra obtained in cross-polarized modes indicate that β-sheets within the SRT are randomly oriented. Radial integration pattern of intensities of (B) SAXS and (C) WAXS experiments performed on native SRT. (D) Schematic illustration of thermally induced transformations at the molecular (bottom) and nano-scale (top), as observed from WAXS and SAXS respectively. (E) Schematic illustration of molecular and nano-scale structure of native, reconstituted and stretched SRT. Panel A reproduced from Ref. [10] with authorization from Nature Publishing Group (Copyright 2013 Nature Publishing Group). Panels B, C and D reproduced from Ref. [24] with authorization from Nature Publishing Group (Copyright 2015 Nature Publishing Group). Panel E reproduced from Ref. [25]. Figure 5. Thermal processing and extrusion of filaments from native SRT. (A) Native SRT were grounded into powder form, then mixed with plasticizing agents (50% SRT, 25% water and 25% glycerol by weight) and heated to 150°C. (B) A 1.5 mm diameter feed filament was vacuum formed from the molten SRT mixture, which could then be (D) thermally extruded into a 300 μm diameter filament through a fused filament fabrication print head attached to a robotic arm (C). Panels reproduced from Ref. [24] with authorization from Nature Publishing Group (Copyright 2015 Nature Publishing Group). ACS Paragon Plus Environment

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Figure 6. Crosslinked suckerin-19 hydrogels with tunable stiffness. (A) Photographs of crosslinked suckerin-19 gels under normal light (top) and UV light (bottom), which illustrates the fluorescence arising from the formation of di-Tyr crosslinks. Spatial presentation of β-sheet strands, with (B) “β-sheet locking” present under moderate di-Tyr crosslinking density condition and (C) high di-Tyr crosslinking density, resulting in constrained flexibility of amorphous domains and eventually to “frustration” of β-sheet formation. (D) Tissue elasticity of a range of biological tissues in comparison with the achieved elastic moduli of processed suckerin-19. Panels reproduced from Ref. [68] with authorization from John Wiley and Sons (Copyright 2015 John Wiley and Sons).

Figure 7. Growth of Au nanoparticles promoted by suckerins. (A) SEM and (B) TEM images of triangular or truncated triangular-shaped Au nanoparticles formed by the addition of suckerin19 protein solution to a solution of HAuCl4. (C) SEM images of fabricated suckerin-12 nanotubes coated with Au nanoparticles, with EDX (inset) validating the presence of gold on the nanotubes’ surface. (D) Illustration of the nanotubular structure fabrication procedure, followed by coating with Au nanoparticles. (E) UV-vis spectra of suckerin-12 film with the growth of Au nanoparticles and an image of the protein film on a glass slide (inset). (F) TEM image of a suckerin-12 film covered with Au nanoparticles and the corresponding SAED pattern (inset). Panels reproduced from Ref. [73] with authorization from John Wiley and Sons (Copyright 2015 John Wiley and Sons).

Figure 8. Suckerin peptide hydrogel formed by self-assembly in water. (A) Photo of a hydrogel formed by suckerin peptide. (B) SEM image of hydrogel’s porous network, with inset illustrating the fibrillar structures forming the gel. (C) Rheological measurements of peptide hydrogel showing the storage modulus (G’) and the loss modulus (G”) at a constant strain of 0.05% over a range of frequency. The inset shows the amplitude sweep where the linear viscoelastic region was obtained, and the selected strain for frequency sweep measurements.


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For Table of Contents Use Only

Squid Sucker Ring Teeth: Multi-Scale Structure-Property Relationships, Sequencing, and Protein Engineering of a Thermoplastic Biopolymer Shu Hui Hiew and Ali Miserez


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Figure 1. Drawing of Humboldt squid (Dosidicus gigas) and its “hard tissues” appendages by French naturalist, Alcide D’Orbigny. (Voyage dans l’Amerique Meridionale: Tome Neuvieme. 1847.) 150x217mm (300 x 300 DPI)



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Figure 2. Mechanical properties and structure of Humboldt squid sucker ring teeth. Scanning electron microscope (SEM) image showing (A) the nano-tubular structure of a SRT fracture section and (B) the porous tubular cross-section of the teeth with inset presenting a scanning probe microscope image of the SRT’s porous cross-section using a nanoindentor in the scanning mode. (C) Materials selection chart (Ashby plot of biological materials with SRT core and periphery indicated, and. (D) Ashby plot of engineering materials such as polymers, metals and ceramic materials, with dried native squid sucker ring teeth (values ranging from core to periphery) indicated. (E) Tensile test curves performed on melt-cast processed SRT ribbons at different humidities, revealing ductile-to-brittle transition from wet to dry samples. Panel C reproduced from Ref. [23] with authorization from Elsevier (Copyright 2013 Elsevier). Panel D reproduced from Ref. [10] with authorization from Nature Publishing Group (Copyright 2013 Nature Publishing Group). Panel E reproduced from Ref. [25]. 199x189mm (300 x 300 DPI)


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Figure 3. SRT and their constitutive suckerins from various cephalopod species. (A) Scalar comparison of three cephalopod species, Humboldt squid (D. gigas)- top, Bigfin reef squid (S. lessoniana)- middle and Golden cuttlefish (S. esulenta)- bottom, and their respective SRT (B). (C) Modular architecture of a representative Humboldt squid suckerin (suckerin-12) showing regular Pro placement, flanking [M1] modules. (D) Distribution of the number of amino acid residues between consecutive Pro for all Humboldt squid suckerins. (E) Phylogenetic tree depicting relationships of all known suckerin proteins, with the classification of six clades. All panels reproduced from Ref. [31] with authorization from American Chemical Society (Copyright 2014 American Chemical Society). 199x90mm (300 x 300 DPI)



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Figure 4. Molecular design and nano-scale structure of SRT. (A) Raman spectra obtained in cross-polarized modes indicate that β-sheets within the SRT are randomly oriented. Radial integration pattern of intensities of (B) SAXS and (C) WAXS experiments performed on native SRT. (D) Schematic illustration of thermally induced transformations at the molecular (bottom) and nano-scale (top), as observed from WAXS and SAXS respectively. (E) Schematic illustration of molecular and nano-scale structure of native, reconstituted and stretched SRT. Panel A reproduced from Ref. [10] with authorization from Nature Publishing Group (Copyright 2013 Nature Publishing Group). Panels B, C and D reproduced from Ref. [24] with authorization from Nature Publishing Group (Copyright 2015 Nature Publishing Group). Panel E reproduced from Ref. [25]. 139x199mm (300 x 300 DPI)


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Figure 5. Thermal processing and extrusion of filaments from native SRT. (A) Native SRT were grounded into powder form, then mixed with plasticizing agents (50% SRT, 25% water and 25% glycerol by weight) and heated to 150°C. (B) A 1.5 mm diameter feed filament was vacuum formed from the molten SRT mixture, which could then be (D) thermally extruded into a 300 µm diameter filament through a fused filament fabrication print head attached to a robotic arm (C). Panels reproduced from Ref. [23] with authorization from Nature Publishing Group (Copyright 2015 Nature Publishing Group). 79x56mm (300 x 300 DPI)



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Figure 6. Crosslinked suckerin-19 hydrogels with tunable stiffness. (A) Photographs of crosslinked suckerin19 gels under normal light (top) and UV light (bottom), which illustrates the fluorescence arising from the formation of di-Tyr crosslinks. Spatial presentation of β-sheet strands, with (B) “β-sheet locking” present under moderate di-Tyr crosslinking density condition and (C) high di-Tyr crosslinking density, resulting in constrained flexibility of amorphous domains and eventually to “frustration” of β-sheet formation. (D) Tissue elasticity of a range of biological tissues in comparison with the achieved elastic moduli of processed suckerin-19. Panels reproduced from Ref. [68] with authorization from John Wiley and Sons (Copyright 2015 John Wiley and Sons). 199x105mm (300 x 300 DPI)


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Figure 7. Growth of Au nanoparticles promoted by suckerins. (A) SEM and (B) TEM images of triangular or truncated triangular-shaped Au nanoparticles formed by the addition of suckerin-19 protein solution to a solution of HAuCl4. (C) SEM images of fabricated suckerin-12 nanotubes coated with Au nanoparticles, with EDX (inset) validating the presence of gold on the nanotubes’ surface. (D) Illustration of the nanotubular structure fabrication procedure, followed by coating with Au nanoparticles. (E) UV-vis spectra of suckerin-12 film with the growth of Au nanoparticles and an image of the protein film on a glass slide (inset). (F) TEM image of a suckerin-12 film covered with Au nanoparticles and the corresponding SAED pattern (inset). Panels reproduced from Ref. [73] with authorization from John Wiley and Sons (Copyright 2015 John Wiley and Sons). 199x113mm (300 x 300 DPI)



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Figure 8. Suckerin peptide hydrogel formed by self-assembly in water. (A) Photo of a hydrogel formed by suckerin peptide. (B) SEM image of hydrogel’s porous network, with inset illustrating the fibrillar structures forming the gel. (C) Rheological measurements of peptide hydrogel showing the storage modulus (G’) and the loss modulus (G”) at a constant strain of 0.05% over a range of frequency. The inset shows the amplitude sweep where the linear viscoelastic region was obtained, and the selected strain for frequency sweep measurements. 199x63mm (300 x 300 DPI)


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Squid Sucker Ring Teeth - American Chemical Society

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