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• W4224452023 abstract "•Identification of a low-complexity protein present in a crosslinked natural system•In vitro characterization of multipole protein functions•Metal-induced phase separation•Interfacial assembly, crosslinking, and thin film formation allows for fiber drawing High-performance natural materials play a critical role in informing modern material design and development. Bloodworm jaws, used for hunting and defense, are lightweight but with impressive impact and wear properties. The jaw composition is exotic at ∼10% copper and a blend of equal parts of protein and melanin, a pigment rarely used in load-bearing functions. Much of jaw material production is dependent on the properties of the protein, which include binding copper, phase separation, catalyzing melanin formation, and assembling the protein-copper-melanin blend. For effective technological translation, research needs to scrutinize the mechanisms of each of these properties and how they relate to one another. Copper binding underpins the formation of a dense protein liquid phase and enables melanin synthesis but later provides self-healing cohesive bridges between the proteins. The research has the potential to change the way we design, make, use, and dispose of our materials. Bloodworm jaws are composites of protein, melanin, and both mineral and ionic copper. To date, nanomechanical tests have correlated the ionic copper and melanin with hardness and wear resistance, but the function of protein is uncertain. Here, we characterize a Gly- and His-rich protein called multi-tasking protein (MTP) and, using recombinant protein, show that it performs six distinct functions critical for jaw formation and performance, namely (1) recruiting 22 equiv of Cu2+, (2) mediating a liquid-liquid phase separation of the MTP-copper complex, (3) catalyzing the oxidation of 3,4-dihydroxyphenylalanine (Dopa) to melanin, (4) templating the interfacial polymerization of melanin, (5) integrating melanin and itself into thin films and fibers, and (6) providing intermolecular cohesion through Cu bridging. MTP achieves all these by assuming unprecedented roles as a building block, organizer, and fabricator—a processing feat of considerable relevance to the autonomous production of other polymer composites, blends, and/or networks. Bloodworm jaws are composites of protein, melanin, and both mineral and ionic copper. To date, nanomechanical tests have correlated the ionic copper and melanin with hardness and wear resistance, but the function of protein is uncertain. Here, we characterize a Gly- and His-rich protein called multi-tasking protein (MTP) and, using recombinant protein, show that it performs six distinct functions critical for jaw formation and performance, namely (1) recruiting 22 equiv of Cu2+, (2) mediating a liquid-liquid phase separation of the MTP-copper complex, (3) catalyzing the oxidation of 3,4-dihydroxyphenylalanine (Dopa) to melanin, (4) templating the interfacial polymerization of melanin, (5) integrating melanin and itself into thin films and fibers, and (6) providing intermolecular cohesion through Cu bridging. MTP achieves all these by assuming unprecedented roles as a building block, organizer, and fabricator—a processing feat of considerable relevance to the autonomous production of other polymer composites, blends, and/or networks. Synthetic hydrogels made from multiple polymer networks are renowned for their strength and toughness.1Dragan E.S. Design and applications of interpenetrating polymer network hydrogels.A. Review. Chem. Eng. J. 2014; 243: 572-590https://doi.org/10.1016/j.cej.2014.01.065Crossref Scopus (655) Google Scholar,2Fan H. Gong J.P. Fabrication of bioinspired hydrogels: challenges and opportunities.Macromolecules. 2020; 53: 2769-2782https://doi.org/10.1021/acs.macromol.0c00238Crossref Scopus (96) Google Scholar The same is true for anhydrous double polymer networks, although fabrication of these remains challenging.3Ducrot E. Chen Y. Bulters M. Sijbesma R.P. Creton C. Toughening elastomers with sacrificial bonds and watching them break.Science. 2014; 344: 186-189https://doi.org/10.1126/science.1248494Crossref PubMed Scopus (703) Google Scholar As living organisms make both hydrogels and less hydrated varieties of tough polymer networks, a detailed study of formation strategies holds many potential insights. For example, vertebrate cartilage is a high-performance load-bearing hydrogel that is formed and maintained by isolated matrix-embedded cells known as chondrocytes that mediate simultaneous co-deposition and turnover of multiple polymer networks.4Halloran J.P. Sibole S. van Donkelaar C.C. van Turnhout M.C. Oomens C.W.J. Weiss J.A. Guilak F. Erdemir A. Multiscale mechanics of articular cartilage: potentials and challenges of coupling musculoskeletal, joint, and microscale computational models.Ann. Biomed. Eng. 2012; 40: 2456-2474https://doi.org/10.1007/s10439-012-0598-0Crossref PubMed Scopus (63) Google Scholar By contrast, in cell-free invertebrate cuticles, such as squid beak, a chitinous hydrogel is deposited first by epidermal cells and then infiltrated with liquid-liquid phase-separated proteins that displace water and harden following oxidation.5Tan Y. Hoon S. Guerette P.A. Wei W. Ghadban A. Hao C. Miserez A. Waite J.H. Infiltration of chitin by protein coacervates defines the squid beak mechanical gradient.Nat. Chem. Biol. 2015; 11: 488-495https://doi.org/10.1038/nchembio.1833Crossref PubMed Scopus (120) Google Scholar Here, we elucidate a third, previously unreported strategy in bloodworm jaws in which a suspension of metallo-protein droplets contributes catalytically and structurally to form a second melanin-like network in situ. The bloodworm Glycera dibranchiata Ehlers, 1868, is a marine polychaete (phylum Annelida) that burrows through intertidal benthic mud to a depth of several meters. The proboscis of each worm is equipped with four black jaws (Figures 1A and 1B ) that grasp and inject venom into other creatures during hunting and combat. The major structural components of Glycera jaws are protein (∼50% w/w), ionic and mineralized copper (up to 10%), and melanin (∼40% w/w).6Lichtenegger H.C. Schöberl T. Bartl M.H. Waite H. Stucky G.D. High abrasion resistance with sparse mineralization: copper biomineral in worm jaws.Science. 2002; 298: 389-392https://doi.org/10.1126/science.1075433Crossref PubMed Scopus (187) Google Scholar,7Moses D.N. Harreld J.H. Stucky G.D. Waite J.H. Melanin and Glycera jaws.J. Biol. Chem. 2006; 281: 34826-34832https://doi.org/10.1074/jbc.M603429200Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar The presence of melanin is not unusual, as melanin is extensively used in biology as a pigment. Jaw melanin, however, is unique in existing as a contiguous material that is ∼2 mm in length, thereby greatly exceeding the micrometer length scale typically associated with dispersions of melanin pigment, such as those found in Sepia ink or melanosomes.8Clancy C.M.R. Simon J.D. Ultrastructural organization of eumelanin from Sepia officinalis measured by atomic force microscopy.Biochemistry. 2001; 40: 13353-13360https://doi.org/10.1021/bi010786tCrossref PubMed Scopus (174) Google Scholar Melanin has many desirable properties,9Hill H.Z. The function of melanin or six blind people examine an elephant.BioEssays. 1992; 14: 49-56https://doi.org/10.1002/bies.950140111Crossref PubMed Scopus (269) Google Scholar but given that in vitro formation typically leads to dispersions of small (∼100 nm diameter) granules,8Clancy C.M.R. Simon J.D. Ultrastructural organization of eumelanin from Sepia officinalis measured by atomic force microscopy.Biochemistry. 2001; 40: 13353-13360https://doi.org/10.1021/bi010786tCrossref PubMed Scopus (174) Google Scholar applications of melanin in synthetic materials remain limited. The melanin and copper in Glycera jaws are correlated with impressive wear resistance,10Pontin M.G. Moses D.N. Waite J.H. Zok F.W. A nonmineralized approach to abrasion-resistant biomaterials.Proc. Natl. Acad. Sci. 2007; 104: 13559-13564https://doi.org/10.1073/pnas.0702034104Crossref PubMed Scopus (43) Google Scholar and a deeper understanding of the mechanisms of their formation and function could lead to expanded use of melanin in high-performance materials. Although previous work has shown that the protein composition of whole jaws is dominated by glycine (Gly) (∼50 mol %) and histidine (His) (∼30 mol %),6Lichtenegger H.C. Schöberl T. Bartl M.H. Waite H. Stucky G.D. High abrasion resistance with sparse mineralization: copper biomineral in worm jaws.Science. 2002; 298: 389-392https://doi.org/10.1126/science.1075433Crossref PubMed Scopus (187) Google Scholar the function of these Gly- and His-rich sequences is currently unknown. Here, we identify and characterize properties of the primary structural protein in bloodworm jaws, hereafter named multi-tasking protein (MTP). Glycera MTP is a multi-functional molecule with the ability to chelate copper, phase-separate, induce the polymerization of L-3,4-dihydroxyphenylalanine (Dopa) to melanin, template the synthesis of macroscopic 2D melanin-protein composite films, and mediate their mechanical properties, all notwithstanding its sequence simplicity. The concerted activities of MTP in the construction of Glycera jaw architecture present a compelling opportunity to rethink the design of processing technologies needed for high-performance and sustainable composite and blended polymeric materials. The everted proboscis of a G. dibranchiata worm in Figure 1A highlights its four black jaws. The jaws are approximately 2 mm in length (Figures 1A and 1B) and have impressive mechanical properties that do not depend on mineralization.10Pontin M.G. Moses D.N. Waite J.H. Zok F.W. A nonmineralized approach to abrasion-resistant biomaterials.Proc. Natl. Acad. Sci. 2007; 104: 13559-13564https://doi.org/10.1073/pnas.0702034104Crossref PubMed Scopus (43) Google Scholar Glycera jaws are highly sclerotized, with protein, copper ions/mineral, and melanin distributed throughout the hierarchical architecture.6Lichtenegger H.C. Schöberl T. Bartl M.H. Waite H. Stucky G.D. High abrasion resistance with sparse mineralization: copper biomineral in worm jaws.Science. 2002; 298: 389-392https://doi.org/10.1126/science.1075433Crossref PubMed Scopus (187) Google Scholar,7Moses D.N. Harreld J.H. Stucky G.D. Waite J.H. Melanin and Glycera jaws.J. Biol. Chem. 2006; 281: 34826-34832https://doi.org/10.1074/jbc.M603429200Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar The protein component has a composition dominated by two amino acids: Gly (>50 mol %) and His (>30 mol %) (Figure 1C). Undermined by the high degree of crosslinking in the jaw, protein extractions have consistently resulted in low yields, although sufficient amounts of a 30-kDa protein were obtained to prove that its amino acid composition matched that of the jaw.11Moses Dana Novak Structure, Biochemistry, and Mechanical Properties of Glycera Marine Worm Jaws. University of California, 2007: 3285839Google Scholar From that point on, we have relied on state-of-the-art transcriptomic methods to investigate this peculiar protein. Motivated by similar work on squid beak5Tan Y. Hoon S. Guerette P.A. Wei W. Ghadban A. Hao C. Miserez A. Waite J.H. Infiltration of chitin by protein coacervates defines the squid beak mechanical gradient.Nat. Chem. Biol. 2015; 11: 488-495https://doi.org/10.1038/nchembio.1833Crossref PubMed Scopus (120) Google Scholar and Nereis jaws,12Broomell C.C. Chase S.F. Laue T. Waite J.H. Cutting edge structural protein from the jaws of Nereis virens.Biomacromolecules. 2008; 9: 1669-1677https://doi.org/10.1021/bm800200aCrossref PubMed Scopus (39) Google Scholar the transcriptome generated from the jaw pulp of G. dibranchiata (the secretory epithelial tissue in contact with the base of the jaw and with histochemical properties similar to those of the jaw13Michel C. Fonze-Vignaux M.T. Voss-Foucart M.F. Données nouvelles sur la morphologie, l’histochimie et la composition chimique des machoires de Glycera convoluta Keferstein (Annélide Polychète).Bull. Biol. Fr. Belg. 1973; 107: 301-321PubMed Google Scholar) provided ∼24,000 transcripts, the most highly represented being cytoskeletal proteins, such as actin and myosin. We identified assembled candidate sequences by searching the transcriptome for transcripts rich in both glycine and histidine. The most compelling candidate sequence is shown in Figure 1D and was chosen because it was a fully assembled transcript, containing a start codon, signal peptide, and stop codon. The predicted protein sequence composition also matched the amino acid composition of jaws (Figure 1C) and showed a similar composition and molecular weight of ∼30 kDa (as determined by SDS-PAGE) to the protein purified from G. dibranchiata jaws in previous work.11Moses Dana Novak Structure, Biochemistry, and Mechanical Properties of Glycera Marine Worm Jaws. University of California, 2007: 3285839Google Scholar We validated the sequence by traditional polymerase chain reaction (PCR) methods, cloning it from fresh cDNA generated from the jaw pulp with primers outside the coding sequence. The PCR product was sequenced by Sanger methods and successfully cloned expressed in Escherichia coli. Consistent with an acid hydrolyzed jaw, the MTP sequence is dominated by Gly and His (over 80%). There are no instances of more than two Gly (G) or His (H) residues in a row, resulting in a sequence made up almost entirely of HGGH, GGH, or HGG repeats, depending on the point of reference. Typical BLAST searches of UniProtKB/Swiss-Prot databases returned no homologous sequences. Not surprisingly, the prevalence of Gly/His in MP does show similarities to that of another polychaete, Nereis virens jaw protein-1 (Nvjp-1),12Broomell C.C. Chase S.F. Laue T. Waite J.H. Cutting edge structural protein from the jaws of Nereis virens.Biomacromolecules. 2008; 9: 1669-1677https://doi.org/10.1021/bm800200aCrossref PubMed Scopus (39) Google Scholar,14Voss-Foucart M.-F. Fonze-Vignaux M.-T. Jeuniaux C. Systematic characters of some polychaetes (Annelida) at the level of the chemical composition of the jaws.Biochem. Syst. Ecol. 1973; 1: 119-122https://doi.org/10.1016/0305-1978(73)90025-2Crossref Scopus (23) Google Scholar although Glycera MTP lacks the abundant aromatic residues of Nvjp-1. The unusually high Gly content of MTP suggests flexibility and intrinsic disorder. High Gly content is found in a number of other structural proteins, such as plant cell wall glycine-rich proteins (60%–70%),15Ringli C. Keller B. Ryser U. Glycine-rich proteins as structural components of plant cell walls.Cell. Mol. Life Sci. 2001; 58: 1430-1441https://doi.org/10.1007/PL00000786Crossref PubMed Scopus (152) Google Scholar spider silk fibroin (47.3%),16Xu M. Lewis R.V. Structure of a protein superfiber: spider dragline silk.Proc. Natl. Acad. Sci. U S A. 1990; 87: 7120-7124https://doi.org/10.1073/pnas.87.18.7120Crossref PubMed Scopus (636) Google Scholar collagen (∼33%), and elastin (∼30%).17Brown-Augsburger P. Tisdale C. Broekelmann T. Sloan C. Mecham R.P. Identification of an elastin cross-linking domain that joins three peptide chains.J. Biol. Chem. 1995; 270: 17778-17783https://doi.org/10.1074/jbc.270.30.17778Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar Moreover, MTP adds to a growing number of Gly- and His-rich structural proteins found in invertebrates, including polychaete (Glycera/Nereis) jaws, squid beaks, Hydra nematocyst spines, insect mandibles, and spider fangs, and all are robust structures both with and without added metal ions.18Degtyar E. Harrington M.J. Politi Y. Fratzl P. The mechanical role of metal ions in biogenic protein-based materials.Angew. Chem. Int. Ed. 2014; 53: 12026-12044https://doi.org/10.1002/anie.201404272Crossref PubMed Scopus (193) Google Scholar The influence of pH and copper on MTP structure is shown in Figures S1 and S2. The CD spectrum at pH 5.0 exhibits an ellipticity minimum at 201 nm, and maxima at 195 and 220 nm. Upon increasing the pH to 7.4 the minimum is shifted to 195 nm, and the intensity of the maximum at 220 nm increases. The addition of copper at pH 7.4 does not result in an appreciable change in the CD spectrum. Although the CD spectrum does change with increasing pH, deconvolution of the spectra with the program BeStSel19Micsonai A. Wien F. Kernya L. Lee Y.-H. Goto Y. Réfrégiers M. Kardos J. Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy.Proc. Natl. Acad. Sci. 2015; 112: E3095-E3103https://doi.org/10.1073/pnas.1500851112Crossref PubMed Scopus (984) Google Scholar estimates that all conditions represent a significant amount of random coil (>40%) and β sheet content (>40%). Hence the predicted flexibility is confirmed by CD measurements. Copper binding ability of the MTP was investigated because the jaw contains significant quantities of copper (up to 10% by weight) that is both ionic and mineral. Isothermal titration calorimetry (ITC) confirmed that MTP has impressive copper binding capacity (Figure 2A). These data were fit to an independent model (NanoAnalyze software), which considers each binding site to be equivalent and independent and was chosen because of its simplicity and appropriateness in situations where the exact nature of the coordination is unknown.20Srivastava V.K. Yadav R. Isothermal titration calorimetry.in: Data Processing Handbook for Complex Biological Data Sources. Elsevier, 2019: 125-137https://doi.org/10.1016/B978-0-12-816548-5.00009-5Crossref Scopus (17) Google Scholar We found an apparent equilibrium constant of KITC = 7.1 × 105 M and a binding capacity of N = 22 equiv of copper. A white precipitate was observed to form over time upon addition of excess copper ions. ITC experiments thus support the notion that His contributes to copper binding. Under our experimental conditions, the apparent equilibrium constant of MTP with copper compares well with that of ceruloplasmin,21Zgirski A. Frieden E. Binding of Cu(II) to non-prosthetic sites in ceruloplasmin and bovine serum albumin.J. Inorg. Biochem. 1990; 39: 137-148https://doi.org/10.1016/0162-0134(90)80022-PCrossref PubMed Scopus (62) Google Scholar but is lower than that of other copper transport proteins, such as serum albumins. The more impressive aspect, however, is the copper binding capacity (N = 22). When normalizing this value to protein MW we find MTP binds 1 Cu2+/kDa, whereas ceruloplasmin binds 0.04 Cu2+/kDa (assuming N = 9 Cu2+ and MW = 121 kDa) and binding stoichiometry for BSA is 0.08 Cu2+/kDa (assuming N = 5 Cu2+ and MW = 66 kDa).22Masuoka J. Hegenauer J. Van Dyke B.R. Saltman P. Intrinsic stoichiometric equilibrium constants for the binding of zinc(II) and copper(II) to the high affinity site of serum albumin.J. Biol. Chem. 1993; 268: 21533-21537https://doi.org/10.1016/S0021-9258(20)80574-2Abstract Full Text PDF PubMed Google Scholar To investigate the structure of MP bound to copper, we performed low-temperature X-band continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy of MTP-Cu2+ complex in Tris buffer at pH 7.4. The spectrum reveals three clear low-field parallel lines with g|| = 2.21 and A|| = 520 MHz, with the fourth obscured by the perpendicular portion (Figure 2B). Simulation of the perpendicular region indicates a g value of 2.00. The low-field edge of the perpendicular region also shows at least nine well-resolved super-hyperfine lines separated by 43 MHz (Figure 2B, inset). Although the copper binding properties are entirely predictable from the high histidine content, the coordination mode is not. The “GGH” motifs that make up the vast majority of the protein sequence are reminiscent of Gly/His model peptides that have been studied to mimic the amino terminal copper and nickel motif23Harford C. Sarkar B. Amino terminal Cu(II)- and Ni(II)-Binding (ATCUN) motif of proteins and peptides: metal binding, DNA cleavage, and other properties.Acc. Chem. Res. 1997; 30: 123-130https://doi.org/10.1021/ar9501535Crossref Scopus (429) Google Scholar as well as those synthesized to mimic the active site of Cu,Zn superoxide dismutase.24Casolaro M. Chelli M. Ginanneschi M. Laschi F. Messori L. Muniz-Miranda M. Papini A.M. Kowalik-Jankowska T. Kozłowski H. Spectroscopic and potentiometric study of the SOD mimic system copper(II)/acetyl-l-histidylglycyl-l-histidylglycine.J. Inorg. Biochem. 2002; 89: 181-190https://doi.org/10.1016/S0162-0134(02)00365-3Crossref PubMed Scopus (54) Google Scholar, 25Jancsó A. Paksi Z. Jakab N. Gyurcsik B. Rockenbauer A. Gajda T. 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Varga-Fábián M. Institórisz L. Balázspiri L. Electron spin resonance study of copper(II) complexes of X-glycine and glycyl-X type dipeptides, and related tripeptides. Variation of co-ordination modes with ligand excess and pH in fluid and frozen aqueous solutions.J. Chem. Soc. Dalt. Trans. 1989; : 1925-1932https://doi.org/10.1039/DT9890001925Crossref Scopus (38) Google Scholar,28Peisach J. Blumberg W.E. Structural implications derived from the analysis of electron paramagnetic resonance spectra of natural and artificial copper proteins.Arch. Biochem. Biophys. 1974; 165: 691-708https://doi.org/10.1016/0003-9861(74)90298-7Crossref PubMed Scopus (1202) Google Scholar However, it is unclear how many of these nitrogens are from the backbone or side-chain imidazole groups. Figure 2C posits copper complex geometries most consistent with these data. To provide more insight into the molecular phenomena underlying the metal-ligand interactions and high copper binding capacity of MTP, and to examine a potential mechanical role for these interactions, we performed single-molecule force spectroscopy (SMFS) experiments on MTP-Cu2+ films. SMFS is a powerful approach for investigating the molecular mechanics of macromolecules.29Zha R.H. Delparastan P. Fink T.D. Bauer J. Scheibel T. Messersmith P.B. Universal nanothin silk coatings via controlled spidroin self-assembly.Biomater. Sci. 2019; 7: 683-695https://doi.org/10.1039/C8BM01186ACrossref PubMed Google Scholar, 30Cao Y. Li H. Polyprotein of GB1 is an ideal artificial elastomeric protein.Nat. Mater. 2007; 6: 109-114https://doi.org/10.1038/nmat1825Crossref PubMed Scopus (193) Google Scholar, 31Puchner E.M. Gaub H.E. Force and function: probing proteins with AFM-based force spectroscopy.Curr. Opin. Struct. 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Single-molecule mechanics of catechol-iron coordination bonds.ACS Biomater. Sci. Eng. 2017; 3: 979-989https://doi.org/10.1021/acsbiomaterials.7b00186Crossref PubMed Scopus (58) Google Scholar Here, we used SMFS to study the force-induced rupture of the inter- and intra-molecular His-Cu2+ interactions present in MTP-Cu2+ thin films. Figure 3A shows a schematic illustration of the simplest case of the SMFS experiments, in which atomic force microscopy (AFM) cantilevers were approached onto MTP-Cu2+ films deposited on TiO2 substrates leading to nonspecific adsorption of a single, isolated protein to the cantilever, and then retracted while measuring the deflection of the cantilever. A characteristic feature of the retraction force-extension curves was the presence of multiple sequential dissociation events as indicated by a “sawtooth” pattern (Figure 3B) reminiscent of that observed upon unfolding of globular proteins under stretch.30Cao Y. Li H. 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Liu X.Y. “Nano-fishnet” structure making silk fibers tougher.Adv. Funct. Mater. 2016; 26: 5534-5541https://doi.org/10.1002/adfm.201600813Crossref Scopus (67) Google Scholar, 38Oberhauser A.F. Marszalek P.E. Carrion-Vazquez M. Fernandez J.M. Single protein misfolding events captured by atomic force microscopy.Nat. Struct. Biol. 1999; 6: 1025-1028https://doi.org/10.1038/14907Crossref PubMed Scopus (174) Google Scholar The sawtooth features were not observed in control experiments performed on Cu2+-free MTP films, leading us to conclude that each subpeak in the sawtooth pattern was the result of force-induced protein chain extension culminating in His-Cu2+ bond rupture, which in turn revealed previously unloaded chain segments (the so-called hidden length).34Huang Z. Delparastan P. Burch P. Cheng J. Cao Y. Messersmith P.B. Injectable dynamic covalent hydrogels of boronic acid polymers cross-linked by bioactive plant-derived polyphenols.Biomater. 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Figure 3C shows a histogram of rupture force distribution for His-Cu2+ interactions with an average of 268 ± 67 pN, stronger than typical hydrogen bonding (∼100 pN) but weaker than covalent bonds (1–3 nN), in agreement with previous reports on the strength of metal-ligand and other non-covalent bonds.34Huang Z. Delparastan P. Burch P. Cheng J. Cao Y. Messersmith P.B. Injectable dynamic covalent hydrogels of boronic acid polymers cross-linked by bioactive plant-derived polyphenols.Biomater. Sci. 2018; 6: 2487-2495https://doi.org/10.1039/C8BM00453FCrossref PubMed Scopus (2) Google Scholar,35Li Y. Wen J. Qin M. Cao Y. Ma H. Wang W. Single-molecule mechanics of catechol-iron coordination bonds.ACS Biomater. Sci. Eng. 2017; 3: 979-989https://doi.org/10.1021/acsbiomaterials.7b00186Crossref PubMed Scopus (58) Google Scholar,39Xue Y. Li X. Li H. Zhang W. Quantifying thiol–gold interactions towards the efficient strength control.Nat. 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