Polymerizing Like Mussels Do: Toward Synthetic Mussel Foot Proteins and Resistant Glues

Abstract A novel strategy to generate adhesive protein analogues by enzyme‐induced polymerization of peptides is reported. Peptide polymerization relies on tyrosinase oxidation of tyrosine residues to Dopaquinones, which rapidly form cysteinyldopa‐moieties with free thiols from cysteine residues, thereby linking unimers and generating adhesive polymers. The resulting artificial protein analogues show strong adsorption to different surfaces, even resisting hypersaline conditions. Remarkable adhesion energies of up to 10.9 mJ m−2 are found in single adhesion events and average values are superior to those reported for mussel foot proteins that constitute the gluing interfaces.

Abstract: An ovel strategy to generate adhesive protein analogues by enzyme-induced polymerization of peptides is reported. Peptide polymerization relies on tyrosinase oxidation of tyrosine residues to Dopaquinones,w hich rapidly form cysteinyldopa-moieties with free thiols from cysteine residues, therebyl inking unimers and generating adhesive polymers. The resulting artificial protein analogues show strong adsorption to different surfaces,even resisting hypersaline conditions. Remarkable adhesion energies of up to 10.9 mJ m À2 are found in single adhesion events and average values are superior to those reported for mussel foot proteins that constitute the gluing interfaces.
For decades,m ussel glues have offered inspiration for av ariety of bio-mimetic materials. [1] Progress in understanding the mechanisms of the concerted self-assembly process of mussel foot proteins (mfps) that constitute the adhesion system led to the recognition of l-3,4-dihydroxyphenylalanine (Dopa) as ak ey moiety for adhesion. [2] This triggered ar ich class of Dopa-carrying polymers with remarkable property profiles. [3] Dopa could either be in-built directly into polymers,l eading to instant adhesives or be enzymatically generated on demand by tyrosinase processing of tyrosinebearing precursor polymers. [4] Besides the contribution of Dopa to adhesion, [5] the role for cohesion is increasingly recognized. [6] Thelatter occurs either non-covalently by Fe 3+ cross-linking of Dopa, [7] or via as et of reaction mechanisms leading to covalent Dopa cross-linking. [8] Among those is the tyrosinase-induced Michael-type addition that constitutes an exploitable pathway for the establishment of an ovel polymerization process.D uring formation of byssal threads, polyphenol oxidase (PPO) converts l-Dopa residues in mfps into l-Dopaquinone [9] in ap rocess that is referred to as quinone tanning.C ross-linking occurs by nucleophilic addition of protein side chain functionalities [10] and one of the most effective pathways involves cysteinea ddition to l-Dopaquinones. [11] Resulting cysteinyldopa cross-links are observed in various adhesive protein systems such as Pcfp-1o fPerna canaliculus and Mcfp-6 footprints of Mytilus californianus. [12] Herein we present our study on abstracting the cysteinedopaquinone addition from mussel adhesive systems to polymerize peptides (unimers) via tyrosinase activation. The enzyme-activated polyaddition exploits the formation of cysteinyldopa connectivities and leads to synthetic adhesive protein analogues.
Themfp-1 family constitutes atough and flexible coating of the byssal threads.I nMytilus edulis,t he Mefp-1 sequence contains ah ighly repetitive decamer AKPSYPPTYK ( Figure 1). [13] Hence,p olymerization of this consensus sequence might result in ap olymer that resembles Mefp-1insome aspects.Tocreate aunimer capable of cysteinyldopa polymerization, AKPSYPPTYK was extended C-terminally with Cys via at ri-glycine spacer, resulting in AKPSY5PP-TY9KGGGC (U 2 C ). Owing to the presence of two tyrosines, U 2 C would produce branched or cross-linked polymers.F or ease of analysis,alinear polymer was anticipated by polymerizing au nimer that contains only Ty r9 as Ty r5 was replaced by Ser5 (AKPSS5PPTY9KGGGC,U 1 C ). Ty rosinase occurs almost ubiquitously throughout nature, is commercially available at low cost, and meets requirements for materials science applications. [14] To avoid batch-to-batch variations, [15] the enzymatic reactions were carried out with the active form of ar ecombinantly expressed tyrosinase (Agaricus bisporus polyphenol oxidase isoform 4, AbPPO4). [14,16] The AbPPO4 belongs to the PPO family from mushrooms and proved to practically instantaneously oxidize the tyrosine residues of U 1 C and U 2 C to Dopa and Dopaquinone as confirmed by MALDI-TOF-MS indicating species with + 16 Da and + 14 Da mass differences compared to non-activated unimers (Supporting Information, Figures S1, S3). More importantly,mass spectrometry as acapable tool for polymer characterization [17] proves the rapid forma-tion of polymerization products.M ALDI-TOF-MS shows species reaching up to about 30 kDa for polyU 1 C and about 25 kDa for polyU 2 C ,w hich could be assigned to ad egree of polymerization (DP) of up to 21 ( Figure 2b). GPC analysis of polyU 1 C revealed the formation of low-and high-molecular weight fractions with apparent peak molecular weights of M P, app. % 20 kDa and M P, app. % 530 kDa, respectively.T he fractions are already present after 5-10 min and correspond to DP p,app. % 15 and 260, respectively.SDS PAGE confirmed the rapid formation of species with 10-25 kDa and suggests that the polymerization appears to be completed within 10 min, as no further band shifting is observed (Supporting Information, Figure S5). Theh igh-molecular-weight fraction found by GPC was not resolved in SDS PAGE owing to the molecular weight cut-off of the gel. Interestingly,t he analysis suggests alimitation in primary polymer growth to occur at amolecular weight of about 20-25 kDa. Theh igher molecular weight fraction is presumably aresult of subsequent cross-linking by secondary reactions such as diDopa formation (Supporting Information, Section S5.12).
ForpolyU 2 C rather related results are observed (Supporting Information, Figure S6). Considering the additional dispersity that is caused by branching,SDS PAGE analysis shows less defined, broader bands,which reach 100 kDa, though the main band also appears at about 20-25 kDa.
MALDI-TOF-MS/MS analysis of the U 1 C polymerization mixture confirmed that the growth mechanism is based on cysteinyldopa linkages (Supporting Information, Section S5.11). TheU 1 C dimer species with m/z 2722.27-2726.30 was fragmented and confirmed the presence of ac ysteinyldopa connectivity by showing required yand bfragmentation ions.M oreover,d irect proof was provided by the molecular ions that result from the SÀC b bond cleavage of the cysteinyldopa species.
Thec ross-linking reaction was investigated in am odel dimerization of the monofunctional unimers AKPSS5PP-TY9KGGGS (U 1 S ), that contains one tyrosine but no cysteine,a nd PTF NO2 KGGGC (U 1N C ), which bears cysteine while tyrosine was replaced with p-nitrophenylalanine.T he dimerization of U 1 S and U 1N C occurs rapidly after enzymatic oxidation of U 1 S via cysteinyldopa formation. HPLC kinetics indicated the complete dimerization product formation within about 5min, even at am arginal excess of U 1N C ( Figure 2d).
Interestingly,asecondary reaction pathway occurred leading to the formation of the disulfide bridged symmetric dimers U 1N The presence of redox active partners like Dopa/Dopaquinone is required to generate disulfides,a st he  control reaction of U 1N C + U 1 S without tyrosinase has shown only 2.5 %d isulfide formation after 48 h( Supporting Information, Figure S16). Thiol oxidation to disulfide can be promoted by reduction of Dopaquinone to Dopa, as has been described for the rescue protein Mcfp-6, which restores Dopa functionalities under the oxidizing seawater conditions. [18] Considering this remarkable analogy,i ti sl ikely that the reduction of Dopaquinone leads to the formation of disulfide and Dopa (Figure 2c,reaction ii). Intriguingly,the U 1N C -U 1N C dimer is not ad ead end, since the redox potential of the reduction of disulfides by Dopa is close to that of the oxidation of thiol by Dopaquinone. [18] Thus,r eaction ii remains reversible and U 1N C can be regenerated to undergo the favored formation of cysteinyldopa linked U 1N C -U 1 S dimers and drives the polymerization (reaction iii). Residual disulfide corresponds to the small excess of U 1N C that was used to reach equivalency of effective functional groups.
Them odel system was further employed to illuminate potential secondary cross-linking routes.U pon enzymatic activation of U 1 S in the absence of U 1N C ,n op olymer was detectable by SDS PAGE. This suggests the need for cysteine to enable polymerization and confirms the lower reactivity of e-amino groups of Lys2 and Lys10 (Supporting Information, Figure S7). However, minor amounts of the dimerization product U 1 S -U 1 S were found after 1h reaction time by MALDI-TOF-MS.E SI-LC-MS/MS confirmed the nature of the connectivity to be a5 ,5'-diDopa linkage as directly associated fragment ions were observed (Supporting Information, Section S5.12). None of the y, b, c, and zions or other fragments gave evidence for lysinyldopa links.A pparently, cysteinyldopa groups are less susceptible for further coupling reactions as no subsequent cross-linking of cysteinyldopa linked U 1 S -U 1N C dimers was observed in am odel reaction over 48 h. Conclusively,secondary cross-linking can occur via 5,5'-diDopa formation by unreacted Dopaquinones,w hich proceeds slower than producing the cysteinyldopal inkages. This evidence supports the hypothesis that the polymerization is driven by cysteinyldopa formation to generate primary polymerization products.T hese can further cross-link via secondary reactions to form high molecular weight fractions.
One of the intriguing aspects of the polymerization is the generation of cysteinyldopa functionalities at each repeat unit, which provide catechol structures and promise high surface-binding capabilities.Aquartz-crystal microbalance with dissipation (QCM-D) was used to gain insights into the adsorption behavior as well as coating stability of polyU 1 C and polyU 2 C .B oth mussel foot protein analogues show rapid adsorption from aqueous solutions onto QCM sensors, exposing either alumina or fluoropolymer surfaces ( Figure 3; Supporting Information, Sections S5.13 and S5.14). Multilayer formation occurs and prevents to reach equilibrium, as it is typical for protein adsorption processes. Swelling of the polyU 2 C coating on alumina is indicated by the gradual decrease in Df during buffer rinsing (Supporting Information, Figure S30). Independent of the substrate,all of the coatings almost completely defy extensive rinsing with buffer and saline seawater equivalents (599 mm NaCl). Most impressively,the coatings withstand hypersaline conditions of 4.2 m NaCl as present in water of the Dead Sea, showing negligible mass losses of 1-7 %. Hence,t he coating systems proved notable adhesion and robust cohesion stabilities.
QCM data evaluation, applying the Voight model for viscoelastic films, [19] enables the estimation of areal mass densities of 40 AE 5mgm À2 and 30 AE 4mgm À2 with corresponding layer thicknesses of 34 AE 4nmand 26 AE 2nmfor polyU 1 C and polyU 2 C ,r espectively.U sing the Sauerbrey model [20] for rigid films to calculate adsorbed polymer masses leads to lower areal mass densities and thus suggests ac ertain viscoelasticity of the polymer films (Supporting Information, Table S4). This is supported by the fact that ap ronounced frequency dependence in the Df and DD response is visible for the coatings ( Supporting Information, Figures S27, S35). By comparing the values of the different models it can be deduced that the polyU 2 C films give more rigid coatings,since the deviation between both models is lower than for polyU 1 C coatings.This can be rationalized by the different types of netpoints in the coatings:while polyU 1 C shows only non-covalent interchain contacts,t he branched topology of polyU 2 C has instead covalent net-points that increase network rigidity. This is consistent with covalently cross-linked Mefp-1 proteins that form more rigid coatings,t hat better suit the Sauerbrey regime. [21] Asimilar trend for coatings of polyU 1 C and polyU 2 C with respect to masses and thicknesses on alumina surfaces is evident for coatings on fluoropolymer substrates (Supporting Information, Figure S43). Thel atter are known to be highly challenging for coating,onwhich nonetheless marine mussels can effectively adhere well. [22] Estimated areal mass densities of 31 AE 3mgm À2 and 18 AE 1mgm À2 (Voight model) give theoretical layer thicknesses of 26 AE 2nma nd 15 AE 1nmf or polyU 1 C and polyU 2 C ,r espectively.T he mass deposition kinetics onto fluoropolymer substrates are initially slightly faster than on alumina, but after 5hof coating time the final areal mass density is lower. This can be correlated to the priming step in the film formation process.Depending on the surface,t he priming involves different contacts between peptide and substrate,w hich will have an influence on follow-up multilayer deposition.
Ultimately,Q CM-D experiments revealed that significantly different surfaces could be effectively coated, leading to stable coatings tolerant to harsh conditions.B oth the cysteinyldopa connectivities and the repetitive mfp-1 consensus sequence were probably synergistically contributing to the properties,a st he unimers and the enzyme alone led to negligible adsorption.
After confirming the exceptional coating behavior of the mussel foot protein analogues,the wet adhesive performance of polyU 1 C and polyU 2 C was investigated with colloidal probe atomic force microscopy (CP-AFM). PolyU 1 C and polyU 2 C were adsorbed onto passivated silicon wafers for 1hand 2h, respectively.A dhesive interactions of the coatings were quantified in force vs.distance measurements with aspherical silica probe of 2.4 mmr adius.T he probe was approached to the coated substrates,v arying the maximum load (2-50 nN) and resting dwell times (0-60 s; Supporting Information, Figure S48). With increasing dwell times,the adhesion of the probe increases in an on-linear fashion for the entire sample set (Supporting Information, Figure S49;F igure 3b). This correlates with ad ynamic character of the interface in the contact area. Both coating polymers will rearrange during contact, thereby optimizing adhesive interactions with the probe.A st he linear polyU 1 C offers higher mobility,w ork of adhesion at 60 sd well time reaches higher values of 1.4 mJ m À2 ,c ompared to 0.5 mJ m À2 for the branched and less flexible polyU 2 C (Figure 3b (2 h);S upporting Information, Figure S49). Theadhesion can be increased significantly to 3.6 mJ m À2 and 0.9 mJ m À2 at 60 sd well time by applying sodium ascorbate antioxidant to the polymer coatings of polyU 1 C and polyU 2 C .T his suggests that oxidation of cysteinyldopa to quinone derivatives might occur during sample preparation and measurements,w here the presence of antioxidants can regenerate the dopa functionalities.T he highest single value that was observed under such conditions reached 10.9 mJ m À2 for polyU 1 C coatings at 30 sdwell time and 20 nN load.
As expected, complex protein-based adhesives like the native mfps exhibit individual surface binding behavior and adaptation dynamics.F or instance,s ilica adhesion of mfp-1, which is related to the synthetic polyU 1 C ,s howed only low work of adhesion of 0.1 mJ m À2 at pH 5.5. [23] It is remarkable that the artificial mfps provide considerably higher work of adhesion. Moreover,t he specialized mfp-3 and mfp-5 that constitute the adhesion interface of mussels,r eached maximum adhesive energy values at pH 5.5 on silica of 2.99 and 2.44 mJ m À2 with ac ontact time of 60 min, [23] respectively, which are still below the average work of adhesion attained by polyU 1 C after only 60 sofcontact time.Ithas also to be noted that ac omplete loss of adhesive function was reported at pH 7-7.5 [22] for several mfps,i ncluding mfp-1, whereas the artificial mfp analogues are able to perform at pH 6.8.
Since there is only limited adhesion data for native mfps and absolute comparison is often difficult, CP-AFM reference experiments with ac ommercially available protein extract from Mytilus edulis (Cell-Tak) were performed. Thec ontrol experiments were carried out under the same conditions as for polyU 1 C (Supporting Information, Figure S50). CP-AFM revealed adhesion energies in asimilar range,with an average of 3.9 mJ m À2 after 60 sdwell time and addition of ascorbate at 2nNl oad force.C onsidering that Cell-Tak is an adhesive optimized for neutral pH, which consists of amixture of mfps, wherein Mefp-1 is one component, and isolated Mefp-1s howed the lowest adhesion properties in previous studies, [24] the polyU 1 C performs very well. Thea rtificial mfp analogue seems to constitute ac apable mfp-1 mimic by just relying on the consensus decapeptide of Mefp-1, but still leaves room for further improvements by sequence adaptation.
In conclusion, anovel tyrosinase-activated polymerization route to mussel foot protein (mfp) analogues was introduced. Polymerization of peptide-based unimers could be achieved using cysteinyldopa linking of an analogue of the Mefp-1consensus decapeptide.Within afew minutes,polymers with an apparent molecular weight of up to 530 kDa are generated. Ther esulting artificial mfp analogues show versatile adsorption and result in coatings having excellent resistance to harsh hypersaline conditions,a sd emonstrated by QCM-D measurements.The adhesive energies in thin-film CP-AFM studies on silica surfaces are superior to values reported from surface apparatus studies on isolated mfp-1 as well as on isolated gluing interface proteins (mfp-3 and mfp-5). Moreover, similar adhesive energy ranges are achieved as with ac ommercial mussel foot protein extract from Mytilus edulis,which is optimized for adhesion at neutral pH. Thet yrosinaseinduced polymerization of peptides offers facile access to artificial mfp analogues and avoids the complexity of naturally derived proteins.N ext generation universal glues can be envisioned that perform effectively even under rigorous seawater conditions and adapt to ab road range of difficult surfaces.