title: Unravelling the Toolbox of Mussel Underwater Adhesion
Creators: name: Wang, Jia
Publisher: name: University of Bayreuth
Year: 2020
Subjects: subject: Mussel foot protein-3b
subject: Recombinant production
subject: Adhesion
subject: Coacervation
subject: Upper critical solution temperature
subject: Polyphenol oxidase like protein
subject: Antioxidant
subject: DOPA
subject: Thiols
subject: 500

description: Over the past decades, biomimetics of novel advanced materials have been developed by replication of natural systems. With respect to tissue adhesives, marine mussel is an ideal model due to its exceptional attachment to substrates in seawater. Mussel-inspired materials are expected to bring about improvements in adhesive technology. A prerequisite for successful biomimetics is to scrutinize the natural system and get fundamental understandings of the adhesion. The attachment of several marine mussels is mediated by a particular holdfast system, the mussel byssus, which is composed of ∼96 % (w/w) of proteins by dry weight. The byssus consists of dozens of byssal threads, which are anchored to the substratum by an adhesive plaque. Mussel foot proteins in plaques are directly contacting the substrates and are, therefore, vital for achieving strong byssus attachment. In this work, mussel foot protein 3b (mfp-3b), one of the predominant proteins in plaques from Mytilus galloprovincialis (M. galloprovincialis) has been recombinantly produced and characterized. rmfp-3b possesses 18 positively and 2 negatively charged residues, which give rise to a soluble state at low pH (pH 3.0-4.0). In the presence of citrate, rmfp-3b showed upper critical solution temperature (UCST) mediated complex coacervation at pH 3.0. Upon decreased temperature, the rmfp-3b solution changed from transparent (T &gt; Tcp, cloud point temperature) to turbid (T &lt; Tcp), and this behavior was reversible. Low temperatures probably can reduce the surrounding water shell of rmfp-3b but increase the hydrogen bonding as well as the amount of deprotonated citrate, which results in increased molecular interactions. Small amounts of multivalent anions gave rise to prominent coacervation, reminiscing that mfp-3b is probably secreted in the form of a condensed coacervate with multivalent anions, e.g. sulfate, in secretory granules. Upon elevation of pH from secretion level (pH 3.0) to seawater level (pH 8.0), mfp-3b coacervate was formed likely due to the deprotonation of acidic residues yielding increased intermolecular interactions, suggesting seawater could be a natural regulator of this process. By means of coacervation, secretion and spreading on wet substrates are beneficial due to the specific properties, such as shear-thinning behavior and low interfacial energy. Thereby, mfp-3b can adhere to wet substrates without dispersion in the surrounding seawater. The high content of 3,4-dihydroxyphenylalanine (Dopa) in byssus indicates the existence of polyphenol oxidases for post-translational modification of Tyr residues, which is pivotal for underwater adhesion. The gene sequence of a polyphenol oxidase like protein (PPOL) was identified after screening of a foot cDNA library of M. galloprovincialis. Recombinant production of PPOL (rPPOL) and the short variant rP319 have been established. The catalytic activity of rPPOL and rP319 have not been identified, which might be due to the poor copper coordination of His residues at acidic pH. rPPOL (rP319) is composed of 15 (5) Cys residues, and ∼13 (∼3) thereof show free thiols and ∼2 (∼2) are disulfide bonded. rPPOL and rP319 were capable of inhibiting Dopa oxidation, whereas alkylated variants showed weaker inhibition, suggesting that PPOL is probably a free thiol-based antioxidant. rPPOL (rP319-NEM) with the most (least) thiols showed maximum (minimum) antiradical power. One molecule rPPOL can reduce ∼38 DPPH radicals, indicating that other reactive groups might exist besides the ∼13 free thiols devoting to the antioxidant activity. Considering the antioxidant property, PPOL probably locates at the plaque-substrate interface to protect Dopa from oxidation, like mfp-6; however, verification of the location of PPOL will need further investigations. This work provides novel insights into the molecular behavior of mfp-3b and PPOL involved in underwater adhesion of marine mussels, which enrich the mechanisms of mussel underwater adhesion and advances future development of tissue adhesives.