Team:KCL UK/MFP Applications

Mussel Foot Proteins

What are mussel foot proteins (MFPs) and what are their biomedical applications?


Figure 1: Illustration of a Mussel.

Mussels spend most of their lives attached to surfaces in turbulent coastal conditions (Waite, 2017). Over time, mussels have evolved a strategy for strong, underwater adhesion to surfaces (Anand and Vardhanan et al., 2020). The byssus (thread) which mussels use to adhere to surfaces form within the mussel foot. The foot emerges and projects outwards towards a surface; by raising the mussel foot, a negative pressure is produced, forming the initial attachment and aiding the secretion of specialised mussel proteins called mussel foot proteins (MFP's). MFP’s have a unique ability to adhere to a variety of surfaces, including those of plastics, metals and rocks in aqueous environments (Waite, 2017). Current adhesives fail to adhere in aqueous conditions, limiting their medical applications. The ability of mussel foot proteins to adhere when wet has garnered the interest of biotechnologists and material scientists, as well as our team.

MFP Adhesion Mechanism:

MFP’s contain an abundance of 3,4-dihydroxyphenylalanine (DOPA), a catechol amino acid modified from tyrosine. These residues make MFP’s "sticky". These proteins, along with many different variants of collagen which make up the core of the thread, are injected into the mussel foot, similar to how we make plastic products using injection moulding. DOPA residues of MFP-5 and MFP-3 form a variety of different bonds dependent on the surface, the most abundant being hydrogen bonding between DOPA and the surface. The variety of bonding that MFP’s are capable of enables mussels to adhere to a range of different surfaces (Waite, 2017).

Pvfp-5β has such immense adhesive properties due to its high concentration of DOPA residues which are able to form adhesive bonds through hydrogen bonding, coordination bonds and metal complexation. Adhesion, however, is not solely due to the presence of DOPA residues, other amino acid side chains within Pvfp-5β can also form hydrogen bonds, electrostatic attraction, cation-π interactions and π-π stacking; all of which aids the adhesion of the mussel byssus to a range of substrate surfaces (Lu et al., 2012). Furthermore, the magnitude of adhesion can also be influenced by the number of basic, aromatic and hydrophobic side chains as well as the spacing between the two hydroxyl groups on DOPA and chain flexibility (Bilotto et al., 2019).

Why employ Pvfp-5β?

There are a variety of different MFP variants across different mussel species. Most mussels utilise six different MFP proteins, all of which are specialised for a specific function in the formation of the mussel byssus. After identifying MFP-5 as the main adhesive protein in most mussel species, we researched different MFP-5 variants from a range of different species. After this research, we decided to incorporate Pvfp-5β into our project. Pvfp-5β is an isoform of pvfp-5 (Perna viridis foot protein), the first MFP to be secreted into the mussel foot in the formation of the mussel byssus in Perva viridis, the asian green mussel. It has significantly greater adhesive properties than its MFP counterparts due to its abundance of DOPA residues, positioned to maximise surface contact and its highly unordered and elongated structure (Petrone et al., 2015).

On top of the adhesive properties of MFP’s, research has identified MFP’s as biocompatible, showing minimal immune reactions in the body. By coating our scaffold in MFP, we hoped to negate the need for immunosuppressant therapy in treatments. As well as this, the presence of MFP’s have been shown to improve cell growth and proliferation, which we hope will improve axon propagation by giving propagating axons a surface to adhere to. They have also been successfully synthesised in recombinant E. coli, ensuring production of our protein is feasible (Santonocito et al., 2019). An MFP-bioadhesive coated scaffold aimed at treating SCI has not yet been created. The aim of our project is to create a novel MFP based bioadhesive for scaffolds in SCI.

Why is auto-oxidation problematic?

In nature, MFPs are co-secreted alongside hydrogen ions which reduces the pH of the mussel foot; this is an adaptation that preserves the chemical structure of the DOPA residues by reducing the likelihood of DOPA oxidation, thus allowing the formation of coordinate bonds and hydrogen bonds. Exposure of MFP-5 to a neutral pH results in >95% decrease in adhesion due to the oxidation of DOPA to DOPA-quinone, resulting in a loss of coordinate and hydrogen bonds (Kan Y, et al.,2014). This is problematic for our project, as implanting an MFP-based bioadhesive coated scaffold into the human body at physiological pH will result in the rapid oxidation of DOPA and a loss of adhesiveness of the bioadhesive polymer. Furthermore, DOPA-quinone encourages cross-linking with other DOPA residues and forms covalent bonds with nucleophilic groups, including -NH2, -SH and imidazole, found on biological substrates - an increase in cross linking results in reduced protein flexibility, a fundamental component of pvfp5β adhesion. In oxidative conditions, reactive oxygen species can form, which can have detrimental effects, leading to a cascade of damage in the site of injury (Forooshani and LEe, 2017). We concluded that the next stage of our research should be looking into protecting DOPA residues via synthetic and biomimetic methods detailed below.

Iron Ion Chelation:

Iron (Fe3+) can form coordinate bonds with hydroxyl groups of dopamine. In doing so, it forms crosslinks between molecules of Pvfp-5β. The complexation of dopamine residues with iron ions aids in resisting their oxidation and acts as a temporary protecting group. However, once complexed, the dopamine is unable to form hydrogen bonds to the substrate as the oxygen anion of the dopamine has bonded to Fe3+. To resolve this, the iron ions must be removed once the mussel foot protein is at its target site (the site of injury in the spine). Deferoxamine (DFO) is a medical iron chelator with an extremely high affinity for Fe3+ ions. DFO could be added in order to compete with the DOPA for Fe3+ ions and release the DOPA residues to adhere to the surface. This would recover the 2 hydroxyls groups on dopamine, ready to form hydrogen bonds with the substrate. This method is two step and would protect dopamine from oxidising to DOPA-quinone, therefore losing adhesion, whilst we place the scaffold in the target area (Fullenkamp et al., 2015).

Figure 2: Hydroxyl groups on the dopamine residues of MFP chelate with iron ions. This complexing increases with Fe3+ concentration, impeding the ability of DOPA to form hydrogen bonds with its substrate.

Boronate Complex:

We looked into synthetic approaches to preventing auto-oxidation of DOPA residues such as the use of boronic acids (BAs) which are known to form boronic acid esters with various diol compounds such as catechols in both organic and aqueous media (Nakahata et al., 2014). BAs have previously been used as temporary protecting groups to preserve the reactivity of the catechol side chain (Narkar et al., 2016). DOPA-boronate complex formation occurs in basic environments at pH 9 and dissociates at acidic pHs or in the presence of negatively charged surfaces. Most surfaces are negatively charged at pH 7.5, thus providing a natural “off-switch” for the boronate complex protection system in physiological conditions (Kan et al., 2014). The dissociation of the catechol protecting group will allow the catecholic hydroxyl groups to form adhesive bonds with the surface. This may prove to be problematic as the free boronate may form boronate esters with other diols in the environment - more research into the composition of CSF and microenvironment must be conducted to rule out the possibility of adverse reactions or boronate. Effective DOPA protection and adhesion requires 1mM borate (12mg/kg) which is a micronutrient with a very low toxicity.


Chlorination of catechols has been shown to inhibit oxidation, which allows for interfacial binding to increase. The chlorination of catechols lowers the pKa of phenolic OH groups and lowers their redox potential, as chlorine withdraws electrons from DOPA making conversion to quinone more difficult (Quan et al., 2019). It was also shown that chlorinated DOPA had interactions that were just as strong as DOPA-interface interactions.


In nature, the mussel byssal plaque secretes acids and antioxidant proteins in order to maintain a low pH and prevent the spontaneous oxidation of DOPA residues. However, any dopamine which is oxidised can be reduced back to its original form by MFP-6. The thiols on the abundance of cysteine residues comprising MFP-6 are oxidised to produce a disulfide bridge, therefore releasing 2 hydrogens which undergo nucleophilic attack from the 2 carbonyl groups on DOPA-quinone (Yu et al., 2011)(Waite, 2017). The half reaction for this reaction is: Q + 2Cys-SH → Cys-S-S + QH2

Inherent anti-oxidative properties of MFP's:

Many mussel foot proteins have unique self-reducing properties, which they use as a defence against oxidation within the formation of the byssal thread. This mechanism is similar to the way MFP-6 functions, except it is an inherent feature of the mussel foot protein which occurs within the protein itself. In nature, MFP’s will perform redox reactions between cysteine and DOPA-quinone residues, where cysteine residues donate electrons into the catechol ring. This leads to the formation of Δ-DOPA, a vinyl catechol with the same metal complexation and hydrogen bonding abilities of DOPA. However, Δ-DOPA is much more susceptible to oxidation, and it has been hypothesised that it mainly serves as an electron reservoir to maintain the adhesiveness of the byssal thread, by donating its electrons to MFP-6 and/or DOPA (Waite, 2017). This is a property which we identified as potentially useful in our project. Once stores of cysteine are depleted, MFP’s search for the aid of MFP-6 continues to restore DOPA-quinone to its reduced form. Despite pvfp-5β having enhanced anti-oxidative abilities due to an abundance of cysteine residues (Petrone et al., 2015), due to its role as the primary adhesive in the mussel byssus, it still has a tendency to oxidise excessively around a neutral pH (Fullenkamp et al., 2014).

MFP Polymerisation

Despite efforts to prevent oxidation of DOPA, it is a fundamental part of the formation of the mussel byssus, factoring towards the structural integrity of the byssal thread. This occurs through DOPA-quinone, the oxidised form of DOPA, which can polymerise into chains that the mussel uses to form the mussel byssus. This is shown below. During the formation of the mussel byssus, Fe3+ ions and O2 leak into the mussel foot, initiating the oxidative process and beginning crosslinking. Nearing the end of the formation of the byssal thread, the mussel foot is lifted, leading to an influx of pH 8 seawater and metal ions. This leads to mass oxidation across the byssal thread, solidifying, strengthening and giving it its supporting shape, forming the intermediate between the adhesive surface and the mussel (Waite, 2017).

Figure 3: Conversion to oxidised DOPA occurring in the mussel byssus, allowing for the polymerisation of MFP chains

We understood that we would need to balance the amounts of DOPA and DOPA-quinone in order to balance the adhesiveness and the structure of our protein polymer, and we optimised an in vivo method to do this

After consulting with our supervisors, Dr Pastore and Dr Alfonso, we decided to research the use of tyrosinase to polymerise mussel foot protein. We were keen on this idea as it would add another degree of biomimicry to our project since mushroom tyrosinase is able to mimic the polymerisation process that takes place in nature during sclerotisation and tanning of the mussel byssal complex (Burzio et al., 2000). Tyrosinase is found in multiple organisms and belongs to the polyphenol oxidase family which catalyses the hydroxylation of tyrosine to DOPA and then the oxidation of DOPA to DOPA-quinone. Tyrosinase results in the formation of cysteinyl-DOPA linkages, resulting in polymerisation. This is illustrated in Figure 4.

Figure 4: Formation of cysteinyl-DOPA linkages from Tyrosinase - which results in polyermisation.


Tyrosinase-catalysed polymerisation is a two-step process; firstly, a cross-linking cysteinyl-DOPA bond is formed by the nucleophilic addition of cysteine to dopaquinone. Secondly, further cross-linking occurs due to unreacted DOPA-quinones reacting to form 5-5’ diDOPA. The polymers created are able to effectively coat significantly different surfaces and are tolerant to harsh conditions. Adhesion of the polymers can reach 3.6mJm-2 by applying sodium ascorbate antioxidant to the polymer coating; this would recover the DOPA residues and restore adhesive functionality of DOPA residues (Horsch et al., 2018).

Figure 5: The two step process of polymerisation catalysed by tyrosinase, beginning with the nucleophilic addition of cysteine to dopaquinone. Tyrosine catalyses the o-hydroxylation of monopehnols (step 1) and the oxidation of o-diphenols (step 2).

Iron Chelation

Fe3+ ions can form coordination complexes between each other at a neutral to high pH. On top of protecting DOPA residues from oxidation, metal ions can be used to link DOPA residues together to form an adhesive film cohered together via metal complexes into a film. This is a natural process which mussels utilize in nature; by mimicking this we hoped to find an effective way to polymerise our protein (Waite, 2017). As these complexes are fully reversible, the adhesive layer will show self-healing properties, increasing the life of the adhesive in an area where it must last to ensure sufficient healing time. However, in the figure below, observe that at an acidic pH, Fe3+ ions can undergo a redox reaction with DOPA, converting it into a polymerised form (Fullenkamp et al., 2014). Overtime, our adhesive would diminish its self-healing properties and pose a risk to free DOPA designated to adhesiveness.

Figure 6: Reaction between Fe3+ and DOPA and marine and acidic conditions.

Bioadhesive Polymer

We will polymerise our protein with the co-expression system of tyrosinase and MFP plasmids, and then use His-tag purification to isolate the protein. To reduce any remaining unreacted dopaquinone back to DOPA we will treat the bioadhesive polymer with sodium ascorbate antioxidant-- this will allow us to maximize the adhesive properties of the catechol group of the MFP. To combat the oxidative properties of DOPA, the bioadhesive will be preserved by adding 1M of boric acid to prevent auto-oxidation. Then, before the scaffold is implanted, it will be submerged into the solubilised bioadhesive polymer.

Therapeutic Applications of MFPs

The formation of DOPA from tyrosine yields a robust, yet reversible bond with inorganic surfaces. However, on organic surfaces, the formation of covalent bonds is more prevalent which allows for adhesion. Due to the reversible nature of their bonds, MFPs are an ideal candidate for biocompatible adhesives. Beyond their use as adhesives for wound closure, mussel foot proteins have shown great promise for use in tissue regeneration and substrates for cellular engineering (Kaushik et al., 2015). Bilic et al. utilised the adhesive properties of the MFPs in an injectable surgical sealant, to be employed for the closure of iatrogenic membrane defects. Mehdizadeh et al., also recently established an injectable citrate-based mussel-inspired bioadhesive for the effective treatment of open bleeding wounds, in the shape of strong biodegradable wet-tissue adhesives. This novel mussel-inspired bioadhesive can be used in topical and non-topical applications, in a plethora of surgical disciplines, from tissue grafts to the treatment of hernia and burns.


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