Dopa (l-3,4-dihydroxyphenylalanine) is a key chemical signature of mussel adhesive proteins, but its susceptibility to oxidation has limited mechanistic investigations as well as practical translation to wet adhesion technology. To investigate peptidyl-Dopa oxidation, the highly diverse chemical environment of Dopa in mussel adhesive proteins was simplified to a peptidyl-Dopa analogue, N-acetyl-Dopa ethyl ester. On the basis of cyclic voltammetry and UV–vis spectroscopy, the Dopa oxidation product at neutral to alkaline pH was shown to be α,β-dehydro-Dopa (ΔD), a vinylcatecholic tautomer of Dopa-quinone. ΔD exhibited an adsorption capacity on TiO2 20-fold higher than that of the Dopa homologue in the quartz crystal microbalance. Cyclic voltammetry confirmed the spontaneity of ΔD formation in mussel foot protein 3F at neutral pH that is coupled to a change in protein secondary structure from random coil to β-sheet. A more complete characterization of ΔD reactivity adds a significant new perspective to mussel adhesive chemistry and the design of synthetic bioinspired adhesives.

Adhesive mussel foot proteins (Mfps) rely in part on DOPA (3,4-dihydroxyphenyl-l-alanine) side chains to mediate attachment to mineral surfaces underwater. Oxidation of DOPA to Dopaquinone (Q) effectively abolishes the adsorption of Mfps to these surfaces. The thiol-rich mussel foot protein-6 (Mfp-6) rescues adhesion compromised by adventitious DOPA oxidation by reducing Q back to DOPA. The redox chemistry and kinetics of foot-extracted Mfp-6 were investigated by using a nonspecific chromogenic probe to equilibrate with the redox pool. Foot-extracted Mfp-6 has a reducing capacity of ∼17 e– per protein; half of this comes from the cysteine residues, whereas the other half comes from other constituents, probably a cohort of four or five nonadhesive, redox-active DOPA residues in Mfp-6 with an anodic peak potential ∼500 mV lower than that for oxidation of cysteine to cystine. At higher pH, DOPA redox reversibility is lost possibly due to Q scavenging by Cys thiolates. Analysis by one- and two-dimensional proton nuclear magnetic resonance identified a pronounced β-sheet structure with a hydrophobic core in foot-extracted Mfp-6 protein. The structure endows redox-active side chains in Mfp-6, i.e., cysteine and DOPA, with significant reducing power over a broad pH range, and this power is measurably diminished in recombinant Mfp-6.

We describe robustly anchored triblock copolymers that adopt loop conformations on surfaces and endow them with unprecedented lubricating and antifouling properties. The triblocks have two end blocks with catechol-anchoring groups and a looping poly(ethylene oxide) (PEO) midblock. The loops mediate strong steric repulsion between two mica surfaces. When sheared at constant speeds of ∼2.5 μm/s, the surfaces exhibit an extremely low friction coefficient of ∼0.002–0.004 without any signs of damage up to pressures of ∼2–3 MPa that are close to most biological bearing systems. Moreover, the polymer loops enhance inhibition of cell adhesion and proliferation compared to polymers in the random coil or brush conformations. These results demonstrate that strongly anchored polymer loops are effective for high lubrication and low cell adhesion and represent a promising candidate for the development of specialized high-performance biomedical coatings.Keywords: antifouling; catechol; lubrication; polymer loops; surface forces apparatus;

The role of friction in the functional performance of biomaterial interfaces is widely reckoned to be critical and complicated but poorly understood. To better understand friction forces, we investigated the natural adaptation of the holdfast or byssus of mussels that live in high-energy surf habitats. As the outermost covering of the byssus, the cuticle deserves particular attention for its adaptations to frictional wear under shear. In this study, we coacervated one of three variants of a key cuticular component, mussel foot protein 1, mfp-1 [(1) Mytilus californianus mcfp-1, (2) rmfp-1, and (3) rmfp-1-Dopa], with hyaluronic acid (HA) and investigated the wear protection capabilities of these coacervates to surfaces (mica) during shear. Native mcfp-1/HA coacervates had an intermediate coefficient of friction (μ ∼0.3) but conferred excellent wear protection to mica with no damage from applied loads, F⊥, as high as 300 mN (pressure, P, > 2 MPa). Recombinant rmfp-1/HA coacervates exhibited a comparable coefficient of friction (μ ∼0.3); however, wear protection was significantly inferior (damage at F⊥ > 60 mN) compared with that of native protein coacervates. Wear protection of rmfp-1/HA coacervates increased 5-fold upon addition of the surface adhesive group 3,4-dihydroxyphenylalanine, (Dopa). We propose a Dopa-dependent wear protection mechanism to explain the differences in wear protection between coacervates. Our results reveal a significant untapped potential for coacervates in applications that require adhesion, lubrication, and wear protection. These applications include artificial joints, contact lenses, dental sealants, and hair and skin conditioners.Keywords: adhesion; biomimetic; HA; hyaluronic acid; interface; mcfp-1; Mytilus californianus foot protein 1; wear protection

Mussel foot protein-1 (mfp-1) is an essential constituent of the protective cuticle covering all exposed portions of the byssus (plaque and the thread) that marine mussels use to attach to intertidal rocks. The reversible complexation of Fe3+ by the 3,4-dihydroxyphenylalanine (Dopa) side chains in mfp-1 in Mytilus californianus cuticle is responsible for its high extensibility (120%) as well as its stiffness (2 GPa) due to the formation of sacrificial bonds that help to dissipate energy and avoid accumulation of stresses in the material. We have investigated the interactions between Fe3+ and mfp-1 from two mussel species, M. californianus (Mc) and M. edulis (Me), using both surface sensitive and solution phase techniques. Our results show that although mfp-1 homologues from both species bind Fe3+, mfp-1 (Mc) contains Dopa with two distinct Fe3+-binding tendencies and prefers to form intramolecular complexes with Fe3+. In contrast, mfp-1 (Me) is better adapted to intermolecular Fe3+ binding by Dopa. Addition of Fe3+ did not significantly increase the cohesion energy between the mfp-1 (Mc) films at pH 5.5. However, iron appears to stabilize the cohesive bridging of mfp-1 (Mc) films at the physiologically relevant pH of 7.5, where most other mfps lose their ability to adhere reversibly. Understanding the molecular mechanisms underpinning the capacity of M. californianus cuticle to withstand twice the strain of M. edulis cuticle is important for engineering of tunable strain tolerant composite coatings for biomedical applications.

The robust, proteinaceous egg capsules of marine prosobranch gastropods (genus Busycotypus) exhibit unique biomechanical properties such as high elastic strain recovery and elastic energy dissipation capability. Capsule material possesses long-range extensibility that is fully recoverable and is the result of a secondary structure phase transition from α-helical coiled-coil to extended β-sheet rather than of entropic (rubber) elasticity. We report here the characterization of the precursor proteins that make up this material. Three different proteins have been purified and analyzed, and complete protein sequences deduced from messenger ribonucleic acid (mRNA) transcripts. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy indicate that the proteins are strongly α-helical in solution and primary sequence analysis suggests that these proteins have a propensity to form coiled-coils. This is in agreement with previous wide-angle X-ray scattering (WAXS) and solid-state Raman spectroscopic analysis of mature egg capsules.

The underwater adhesion of marine mussels relies on mussel foot proteins (mfps) rich in the catecholic amino acid 3,4-dihydroxyphenylalanine (Dopa). As a side chain, Dopa is capable of strong bidentate interactions with a variety of surfaces, including many minerals and metal oxides. Titanium is among the most widely used medical implant material and quickly forms a TiO2 passivation layer under physiological conditions. Understanding the binding mechanism of Dopa to TiO2 surfaces is therefore of considerable theoretical and practical interest. Using a surface forces apparatus, we explored the force–distance profiles and adhesion energies of mussel foot protein 3 (mfp-3) to TiO2 surfaces at three different pHs (pH 3, 5.5 and 7.5). At pH 3, mfp-3 showed the strongest adhesion force on TiO2, with an adhesion energy of ∼−7.0 mJ/m2. Increasing the pH gives rise to two opposing effects: (1) increased oxidation of Dopa, thus, decreasing availability for the Dopa-mediated adhesion, and (2) increased bidentate Dopa-Ti coordination, leading to the further stabilization of the Dopa group and, thus, an increase in adhesion force. Both effects were reflected in the resonance-enhanced Raman spectra obtained at the three deposition pHs. The two competing effects give rise to a higher adhesion force of mfp-3 on the TiO2 surface at pH 7.5 than at pH 5.5. Our results suggest that Dopa-containing proteins and synthetic polymers have great potential as coating materials for medical implant materials, particularly if redox activity can be controlled.

Complex coacervates prepared from poly(aspartic acid) (polyAsp) and poly-l-histidine (polyHis) were investigated as models of the metastable protein phases used in the formation of biological structures such as squid beak. When mixed, polyHis and polyAsp form coacervates whereas poly-l-glutamic acid (polyGlu) forms precipitates with polyHis. Layer-by-layer (LbL) structures of polyHis–polyAsp on gold substrates were compared with those of precipitate-forming polyHis–polyGlu by monitoring with iSPR and QCM-D. PolyHis–polyAsp LbL was found to be stiffer than polyHis–polyGlu LbL with most water evicted from the structure but with sufficient interfacial water remaining for molecular rearrangement to occur. This thin layer is believed to be fluid and like preformed coacervate films, capable of spreading over both hydrophilic ethylene glycol as well as hydrophobic monolayers. These results suggest that coacervate-forming polyelectrolytes deserve consideration for potential LbL applications and point to LbL as an important process by which biological materials form.

Dopa (3,4-dihydroxyphenylalanine) is recognized as a key chemical signature of mussel adhesion and has been adopted into diverse synthetic polymer systems. Dopa’s notorious susceptibility to oxidation, however, poses significant challenges to the practical translation of mussel adhesion. Using a surface forces apparatus to investigate the adhesion of mussel foot protein 3 (Mfp3) “slow”, a hydrophobic protein variant of the Mfp3 family in the plaque, we have discovered a subtle molecular strategy correlated with hydrophobicity that appears to compensate for Dopa instability. At pH 3, where Dopa is stable, Mfp3 slow, like Mfp3 “fast” adhesion to mica, is directly proportional to the mol % of Dopa present in the protein. At pH of 5.5 and 7.5, however, loss of adhesion in Mfp3 slow was less than half that occurring in Mfp3 fast, purportedly because Dopa in Mfp3 slow is less prone to oxidation. Indeed, cyclic voltammetry showed that the oxidation potential of Dopa in Mfp3 slow is significantly higher than in Mfp3 fast at pH of 7.5. A much greater difference between the two variants was revealed in the interaction energy of two symmetric Mfp3 slow films (Ead = −3 mJ/m2). This energy corresponds to the energy of protein cohesion which is notable for its reversibility and pH independence. Exploitation of aromatic hydrophobic sequences to protect Dopa against oxidation as well as to mediate hydrophobic and H-bonding interactions between proteins provides new insights for developing effective artificial underwater adhesives.

Marine mussels utilize a variety of DOPA-rich proteins for purposes of underwater adhesion, as well as for creating hard and flexible surface coatings for their tough and stretchy byssal fibers. In the present study, moderately strong, yet reversible wet adhesion between the protective mussel coating protein, mcfp-1, and amorphous titania was measured with a surface force apparatus (SFA). In parallel, resonance Raman spectroscopy was employed to identify the presence of bidentate DOPA–Ti coordination bonds at the TiO2–protein interface, suggesting that catechol–TiO2 complexation contributes to the observed reversible wet adhesion. These results have important implications for the design of protective coatings on TiO2.

The holdfast or byssus of Asian green mussels, Perna viridis, contains a foot protein, pvfp-1, that differs in two respects from all other known adhesive mussel foot proteins (mfp): (1) instead of the hallmark L-3,4-dihydroxyphenylalanine (DOPA) residues in mfp-1, for example, pvfp-1 contains C2-mannosyl-7-hydroxytryptophan (Man7OHTrp). (2) In addition, pvfp-1 chains are not monomeric like mfp-1 but trimerized by collagen and coiled-coil domains near the carboxy terminus after a typical domain of tandemly repeated decapeptides. Here, the contribution of these peculiarities to adhesion was examined using a surface forces apparatus (SFA). Unlike previously studied mfp-1s, pvfp-1 showed significant adhesion to mica and, in symmetric pvfp-1 films, substantial cohesive interactions were present at pH 5.5. The role of Man7OHTrp in adhesion is not clear, and a DOPA-like role for Man7OHTrp in metal complexation (e.g., Cu2+, Fe3+) was not observed. Instead, cation–π interactions with low desolvation penalty between Man7OHTrp and lysyl side chains and conformational changes (raveling and unraveling of collagen helix and coiled-coil domains) are the best explanations for the strong adhesion between pvfp-1 monomolecular films. The strong adhesion mechanism induced by cation–π interactions and conformational changes in pvfp-1 provides new insights for the development of biomimetic underwater adhesives.

Mussels have a remarkable ability to attach their holdfast, or byssus, opportunistically to a variety of substrata that are wet, saline, corroded, and/or fouled by biofilms. Mytilus edulis foot protein-5 (Mefp-5) is one of several proteins in the byssal adhesive plaque of the mussel M. edulis. The high content of 3,4-dihydroxyphenylalanine (Dopa) (∼30 mol %) and its localization near the plaque–substrate interface have often prompted speculation that Mefp-5 plays a key role in adhesion. Using the surface forces apparatus, we show that on mica surfaces Mefp-5 achieves an adhesion energy approaching Ead = ∼−14 mJ/m2. This exceeds the adhesion energy of another interfacial protein, Mefp-3, by a factor of 4–5 and is greater than the adhesion between highly oriented monolayers of biotin and streptavidin. The adhesion to mica is notable for its dependence on Dopa, which is most stable under reducing conditions and acidic pH. Mefp-5 also exhibits strong protein–protein interactions with itself as well as with Mefp-3 from M. edulis.

Metal-containing polymer networks are widespread in biology, particularly for load-bearing exoskeletal biomaterials. Mytilus byssal cuticle is an especially interesting case containing moderate levels of Fe3+ and cuticle protein—mussel foot protein-1 (mfp-1), which has a peculiar combination of high hardness and high extensibility. Mfp-1, containing 13 mol % of dopa (3, 4-dihydroxyphenylalanine) side-chains, is highly positively charged polyelectrolyte (pI ∼ 10) and didn’t show any cohesive tendencies in previous surface forces apparatus (SFA) studies. Here, we show that Fe3+ ions can mediate unusually strong interactions between the positively charged proteins. Using an SFA, Fe3+ was observed to impart robust bridging (Wad ≈ 4.3 mJ/m2) between two noninteracting mfp-1 films in aqueous buffer approaching the ionic strength of seawater. The Fe3+ bridging between the mfp-1-coated surfaces is fully reversible in water, increasing with contact time and iron concentration up to 10 μM; at 100 μM, Fe3+ bridging adhesion is abolished. Bridging is apparently due to the formation of multivalent dopa-iron complexes. Similar Fe-mediated bridging (Wad ≈ 5.7 mJ/m2) by a smaller recombinant dopa-containing analogue indicates that bridging is largely independent of molecular weight and posttranslational modifications other than dopa. The results suggest that dopa-metal interactions may provide an energetic new paradigm for engineering strong, self-healing interactions between polymers under water.

Biomineralization is an intricate process that relies on precise physiological control of solution and interface properties. Despite much research of the process, mechanistic details of biomineralization are only beginning to be understood, and studies of additives seldom investigate a wide space of chemical conditions in mineralizing solutions. We present a ternary diagram-based method that globally identifies the changing roles and effects of polymer additives in mineralization. Simple polyanions were demonstrated to induce a great variety of morphologies, each of which can be selectively and reproducibly fabricated. This chemical and physical analysis also aided in identifying conditions that selectively promote heterogeneous nucleation and controlled cooperative assembly, manifested here in the form of highly organized cones. Similar complex shapes of CaCO3 have previously been synthesized using double hydrophilic block copolymers. We have found the biomimetic mineralization process to occur interfacially and by the assembly of precursor modules, which generate large mesocrystals with high dependence on pH and substrate surface.

For lasting holdfast attachment, the mussel Mytilus californianus coats its byssal threads with a protective cuticle 2−5 μm thick that is 4−6 times stiffer than the underlying collagen fibers. Although cuticle hardness (0.1 GPa) and stiffness (2 GPa) resemble those observed in related mussels, a more effective dispersion of microdamage enables M. californianus byssal threads to sustain strains to almost 120% before cuticle rupture occurs. Underlying factors for the superior damage tolerance of the byssal cuticle were explored in its microarchitecture and in the cuticular protein, mcfp-1. Cuticle microstructure was distinctly granular, with granule diameters (∼200 nm) only a quarter of those in M. galloprovincialis cuticle, for example. Compared with homologous proteins in related mussel species, mcfp-1 from M. californianus had a similar mass (∼92 kDa) and number of tandemly repeated decapeptides, and contained the same post-translational modifications, namely, trans-4-hydroxyproline, trans-2,3-cis-3,4-dihydroxyproline, and 3,4-dihydroxyphenylalanine (Dopa). The prominence of isoleucine in mcfp-1, however, distinguished it from homologues in other species. The complete protein sequence deduced from cDNAs for two related variants revealed a highly conserved consensus decapeptide PKISYPPTYK that is repeated 64 times and differs slightly from the consensus peptide (AKPSYPPTYK) of both M. galloprovincialis and M. edulis proteins.

Several naturally occurring biomacromolecular structures, particularly those containing histidine-rich proteins, have been shown to depend on metal ion complexation for hardness and stiffness. In this study, water-soluble metal-binding polymers and copolymers based on vinylimidazole were utilized to mimic the glycine- and histidine-rich proteins of ragworm jaws. Blends of these polymers with agarose exhibited a significant capacity for Zn(II) and Cu(II) complexation. Rheological and uniaxial tensile tests as well as nanoindentational analysis of the blends revealed a more than 10-fold improvement in the tensile strength, along with increases in the hardness of the dried samples, upon metal ion addition. Pronounced differences in mechanical effects, however, were associated with Cu(II) and Zn(II) complexation, and the latter provided much better overall mechanical performance.

Higher animals typically rely on calcification to harden certain tissues such as bones and teeth. Some notable exceptions can be found in invertebrates: The fangs, teeth, and mandibles of diverse arthropod species have been reported to contain high levels of zinc. Considerable quantities of zinc also occur in the jaws of the marine polychaete worm Nereis sp. High copper levels in the polychaete worm Glycera dibranchiata recently were attributed to a copper-based biomineral reinforcing the jaws. In the present article, we attempt to unravel the role of zinc in Nereis limbata jaws, using a combination of position-resolved state-of-the-art techniques. It is shown that the local hardness and stiffness of the jaws correlate with the local zinc concentration, pointing toward a structural role for zinc. Zinc always is detected in tight correlation with chlorine, suggesting the presence of a zinc–chlorine compound. No crystalline inorganic phase was found, however, and results from x-ray absorption spectroscopy further exclude the presence of simple inorganic zinc–chlorine compounds in amorphous form. The correlation of local histidine levels in the protein matrix and zinc concentration leads us to hypothesize a direct coordination of zinc and chlorine to the protein. A comparison of the role of the transition metals zinc and copper in the jaws of two polychaete worm species Nereis and Glycera, respectively, is presented.

Marine mussels utilize a variety of DOPA-rich proteins for purposes of underwater adhesion, as well as for creating hard and flexible surface coatings for their tough and stretchy byssal fibers. In the present study, moderately strong, yet reversible wet adhesion between the protective mussel coating protein, mcfp-1, and amorphous titania was measured with a surface force apparatus (SFA). In parallel, resonance Raman spectroscopy was employed to identify the presence of bidentate DOPA–Ti coordination bonds at the TiO2–protein interface, suggesting that catechol–TiO2 complexation contributes to the observed reversible wet adhesion. These results have important implications for the design of protective coatings on TiO2.