Mytilus foot protein type 6 (mfp-6) is crucial for maintaining the reducing conditions needed for optimal wet adhesion in marine mussels. In this report, we describe the expression and production of a recombinant Mytilus californianus foot protein type 6 variant 1 (rmfp-6.1) fused with a hexahistidine affinity tag in Escherichia coli and its purification by affinity chromatography. Recombinant mfp-6 showed high purification yields of 5-6 mg L(-1) cell culture and excellent solubility in low pH buffers that retard oxidation of its many thiol groups. Purified rmfp-6.1 protein showed high 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity when compared with vitamin C. Using the highly sensitive surface forces apparatus (SFA) technique to measure interfacial surface forces in the nano-Newton range, we show that rmfp-6.1 is also able to rescue the oxidation-dependent adhesion loss of mussel foot protein 3 (mfp-3) at pH 3. The adhesion rescue is related to a reduction of dopaquinone back to 3,4-dihydroxyphenyl-l-alanine in mfp-3, which is the reverse reaction observed during the detrimental enzymatic browning process in fruits and vegetables. Broadly viewed, rmfp-6.1 has potential as a versatile antioxidant for applications ranging from personal products to antispoilants for perishable foods during processing and storage. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1587-1593, 2013.

Understanding the interactions between collagen and adhesive mussel foot proteins (mfps) can lead to improved medical and dental adhesives, particularly for collagen-rich tissues. Here we investigated interactions between collagen type-1, the most abundant load-bearing animal protein, and mussel foot protein-3 (mfp-3) using a quartz crystal microbalance and surface forces apparatus (SFA). Both hydrophilic and hydrophobic variants of mfp-3 were exploited to probe the nature of the interaction between the protein and collagen. Our chief findings are: 1) mfp-3 is an effective chaperone for tropocollagen adsorption to TiO2 and mica surfaces; 2) at pH 3, collagen addition between two mfp-3 films (Wc = 5.4 ± 0.2 mJ/m(2)) increased their cohesion by nearly 35%; 3) oxidation of Dopa in mfp-3 by periodate did not abolish the adhesion between collagen and mfp-3 films, and 4) collagen bridging between both hydrophilic and hydrophobic mfp-3 variant films is equally robust, suggesting that hydrophobic interactions play a minor role. Extensive H-bonding, π-cation and electrostatic interactions are more plausible to explain the reversible bridging of mfp-3 films by collagen.

Marine organisms process and deliver many of their underwater coatings and adhesives as complex fluids. In marine mussels one such fluid, secreted during the formation of adhesive plaques, consists of a concentrated colloidal suspension of a mussel foot protein (mfp) known as Mfp-3S. The results of this study suggest that Mfp-3S becomes a complex fluid by a liquid-liquid phase separation from equilibrium solution at a pH and ionic strength reminiscent of the conditions created by the mussel foot during plaque formation. The pH dependence of phase separation and its sensitivity indicate that inter-/intra-molecular electrostatic interactions are partially responsible for driving the phase separation. Hydrophobic interactions between the non- polar Mfp-3S proteins provide another important driving force for coacervation. As complex coacervation typically results from charge-charge interactions between polyanions and polycations, Mfp-3S is thus unique in being the only known protein that coacervates with itself. The Mfp-3S coacervate was shown to have an effective interfacial energy of ⩽1mJm(-2), which explains its tendency to spread over or engulf most surfaces. Of particular interest to biomedical applications is the extremely high adsorption capacity of coacervated Mfp-3S on hydroxyapatite.

Mussel (Mytilus californianus) adhesion to marine surfaces involves an intricate and adaptive synergy of molecules and spatio-temporal processes. Although the molecules, such as mussel foot proteins (mfps), are well characterized, deposition details remain vague and speculative. Developing methods for the precise surveillance of conditions that apply during mfp deposition would aid both in understanding mussel adhesion and translating this adhesion into useful technologies. To probe the interfacial pH at which mussels buffer the local environment during mfp deposition, a lipid bilayer with tethered pH-sensitive fluorochromes was assembled on mica. The interfacial pH during foot contact with modified mica ranged from 2.2 to 3.3, which is well below the seawater pH of ~ 8. The acidic pH serves multiple functions: it limits mfp-Dopa oxidation, thereby enabling the catecholic functionalities to adsorb to surface oxides by H-bonding and metal ion coordination, and provides a solubility switch for mfps, most of which aggregate at pH ≥ 7-8.

The 3,4-dihydroxyphenylalanine (Dopa)-containing proteins of marine mussels provide attractive design paradigms for engineering synthetic polymers that can serve as high performance wet adhesives and coatings. Although the role of Dopa in promoting adhesion between mussels and various substrates has been carefully studied, the context by which Dopa mediates a bridging or nonbridging macromolecular adhesion to surfaces is not understood. The distinction is an important one both for a mechanistic appreciation of bioadhesion and for an intelligent translation of bioadhesive concepts to engineered systems. On the basis of mussel foot protein-5 (Mfp-5; length 75 res), we designed three short, simplified peptides (15-17 res) and one relatively long peptide (30 res) into which Dopa was enzymatically incorporated. Peptide adhesion was tested using a surface forces apparatus. Our results show that the short peptides are capable of weak bridging adhesion between two mica surfaces, but this adhesion contrasts with that of full length Mfp-5, in that (1) while still dependent on Dopa, electrostatic contributions are much more prominent, and (2) whereas Dopa surface density remains similar in both, peptide adhesion is an order of magnitude weaker (adhesion energy E(ad) ∼ -0.5 mJ/m(2)) than full length Mfp-5 adhesion. Between two mica surfaces, the magnitude of bridging adhesion was approximately doubled (E(ad) ∼ -1 mJ/m(2)) upon doubling the peptide length. Notably, the short peptides mediate much stronger adhesion (E(ad) ∼ -3.0 mJ/m(2)) between mica and gold surfaces, indicating that a long chain length is less important when different interactions are involved on each of the two surfaces.

Sessile marine mussels must "dry" underwater surfaces before adhering to them. Synthetic adhesives have yet to overcome this fundamental challenge. Previous studies of bioinspired adhesion have largely been performed under applied compressive forces, but such studies are poor predictors of the ability of an adhesive to spontaneously penetrate surface hydration layers. In a force-free approach to measuring molecular-level interaction through surface-water diffusivity, different mussel foot proteins were found to have different abilities to evict hydration layers from surfaces-a necessary step for adsorption and adhesion. It was anticipated that DOPA would mediate dehydration owing to its efficacy in bioinspired wet adhesion. Instead, hydrophobic side chains were found to be a critical component for protein-surface intimacy. This direct measurement of interfacial water dynamics during force-free adsorptive interactions at solid surfaces offers guidance for the engineering of wet adhesives and coatings.

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 Fe(3+) 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 Fe(3+) 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 Fe(3+), mfp-1 (Mc) contains Dopa with two distinct Fe(3+)-binding tendencies and prefers to form intramolecular complexes with Fe(3+). In contrast, mfp-1 (Me) is better adapted to intermolecular Fe(3+) binding by Dopa. Addition of Fe(3+) 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.