ARTICLE

Received 20 Jun 2015 | Accepted 24 Sep 2015 | Published 28 Oct 2015 DOI: 10.1038/ncomms9737 OPEN Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins

Luigi Petrone1,2, Akshita Kumar2,3, Clarinda N. Sutanto1,2, Navinkumar J. Patil4, Srinivasaraghavan Kannan5, Alagappan Palaniappan1,2, Shahrouz Amini1,2, Bruno Zappone6, Chandra Verma3,5,7 & Ali Miserez1,2,3

Interfacial water constitutes a formidable barrier to strong surface bonding, hampering the development of water-resistant synthetic adhesives. Notwithstanding this obstacle, the Asian green mussel Perna viridis attaches firmly to underwater surfaces via a proteinaceous secretion (byssus). Extending beyond the currently known design principles of mussel adhesion, here we elucidate the precise time-regulated secretion of P. viridis mussel adhesive proteins. The vanguard 3,4-dihydroxy-L-phenylalanine (Dopa)-rich protein Pvfp-5 acts as an adhesive primer, overcoming repulsive hydration forces by displacing surface-bound water and generating strong surface adhesion. Using homology modelling and molecular dynamics simulations, we find that all mussel adhesive proteins are largely unordered, with Pvfp-5 adopting a disordered structure and elongated conformation whereby all Dopa residues reside on the protein surface. Time-regulated secretion and structural disorder of mussel adhesive proteins appear essential for optimizing extended nonspecific surface interactions and byssus’ assembly. Our findings reveal molecular-scale principles to help the development of wet-resistant adhesives.

1 School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore. 2 Centre for Biomimetic Sensor Science, Nanyang Technological University, 50 Nanyang Drive, Research Techno Plaza, XFrontiers Block, Singapore 637553, Singapore. 3 School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore. 4 Dipartimento di Fisica, Universita` della Calabria, 87036 Arcavacata di Rende (CS), Italy. 5 Bioinformatics Institute A*Star, 30 Biopolis Street, Singapore 138671, Singapore. 6 Consiglio Nazionale delle Ricerche, CNR-Nanotec, UOS Licryl-Cosenza, 87036 Rende (CS), Italy. 7 Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore. Correspondence and requests for materials should be addressed to A.M. (email: [email protected]).

NATURE COMMUNICATIONS | 6:8737 | DOI: 10.1038/ncomms9737 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9737

ater is regarded as a contaminant in adhesion aimed at preventing mussels from attaching to man-made technology because interfacial water leads to marked underwater structures (for example, ship hulls and drilling Wbond failure1–3. Despite this prevalent challenge, platforms), decreasing frictional drag, fuel consumption and wave-swept rocky shores are home to a variety of sessile maintainance24. organisms that have evolved to attach themselves to submerged surfaces, forming dense communities, such as mussel beds, via self-organizing processes at the individual and the ecosystem Results 4–7 level . Mussels secrete a protein-based holdfast (byssus) Time-resolved secretion of mussel adhesive proteins. The strongly anchoring themselves to underwater solid substrates. injection of KCl solution in the pedal nerve at the base of the The byssus distal end (byssal plaque) is specialized for adhesion, mussel foot induced the secretion of a thread from the groove and six mussel foot proteins have been identified in the within the foot. When a glass slide was placed on top of the foot, Mytilus genus (mfp-2, -3S, -3F, -4, -5 and -6). All mpfs are the freshly secreted thread adhered to the glass via its adhesive post-translationally modified to various extents with the amino plaque. The thread was also elastic and the plaque was firmly 1,8,9 acid 3,4-dihydroxyphenyl-L-alanine (Dopa) . While initial attached to the glass surface, demonstrating that the artificially studies have pointed out the definite role of Dopa in induced secretion is a reasonable mimic of the natural byssus 8–11 mussel adhesion , driving intense research efforts to develop (Fig. 1a–d). Saline-induced adhesive secretions from mussels of Dopa-containing polymers in applications such as wet adhesion P. viridis (n ¼ 15) were collected from the groove at the tip of the promoters, medical sealants, self-healing polymers and anti- foot organ (Supplementary Fig. 1) before the saline injection 12–15 fouling coatings , recent work has demonstrated that the and after injection at times ranging between 10 s and 30 min. success of mussel adhesion goes beyond the ‘Dopa paradigm’. The samples were analysed by matrix-assisted laser desorption/ These studies have notably revealed that redox interactions ionization time-of-flight mass spectrometry (MALDI-ToF MS) 16 between mfps are key to maintaining Dopa adhesive activity , during the course of the secretion. Some differences were that hydrophobic/hydrophilic interactions can also engage in observed between specimens in terms of relative peak intensities 17,18 adhesive interactions or that the local concentration of and number of Pvfp variants. In most cases, a higher intensity adhesive proteins during secretion also plays a critical role to was recorded for Pvfp-5 variants after 10 s, 5 min and 10 min 19 ensure proper plaque delivery . However, one important aspect (Fig. 1e), whereas in a few cases, Pvfp-5 variants were also that has eluded mussel adhesion research so far is the precise observed after 30 min (Supplementary Fig. 2). While there was determination of adhesive proteins’ secondary and tertiary biological variation among specimens (Supplementary Table 1), a structures, which are intrinsically related to their extensive clear trend emerged with Pvfp-5 variants in the 8–10 kDa range nonspecific adsorption. always being secreted first, typically within 10 s after saline 20 Combining RNA-seq with proteomic studies , we have injection and before other Pvfps. Pvfp-3 variants in the 5–6 kDa recently identified the byssal plaque proteins from the Perna range appeared 30 s after injection, often concurrently with genus. Three Dopa-containing foot proteins, termed Pvfp-3, Pvfp-5. Depending on the individual mussel, Pvfp-6 at 11.4 kDa -5 and -6, have been identified in the Asian green mussel Perna appeared from 5 min after injection, along with Pvfp-3 and -5. viridis (P. viridis), whose primary sequences share very low The total mass of Pvfps at each time interval after saline injection 20,21 homology with mfps . Both mfps and Pvfps comprise was determined by Bradford assay, revealing a fast initial polymorphic families, with multiple genes and gene copies secretion rate reaching a total mass of 50–60 mg after ca. 10 min expressing different variants or processing diverse versions of (Fig. 1f). The similarity between artificial and natural secretions mRNA via alternative splicing. Not all variants are eventually was further verified by performing MALDI-Tof MS analysis on a translated and biological variation is often observed among natural mussel’s footprint deposited on glass. The MALDI peaks 1,22 individuals , with Pvfps spanning characteristic molecular matched those from the saline-induced secretion, with Pvfp-3, weight in the range 4.5–6 kDa for Pvfp-3, 8–10.5 and -5 and -6 variants detected (Fig. 1e and Supplementary Table 1). 16–18 kDa for Pvfp-5, and a single protein of 11.4 kDa for Pvfp-6. To fully understand mussel adhesion, it is essential to determine whether adhesive proteins are simultaneously dis- Size and secondary structure of native Pvfps. Pvfp-3a,-5b and charged on surfaces as a complex matrix or deposited in distinct -6 isolated and purified from mussel foot were shown to contain succession, as well as to establish the interplay between adhesive Dopa, as visible in the positive nitroblue tetrazolium (NBT) proteins’ conformations in solution and protein–substrate inter- staining (Supplementary Figs 3 and 4), and their molecular actions. Here we address these two aspects and establish that the weight was determined by MALDI-ToF MS (Supplementary secretion is precisely time regulated. Pvfp-5 is the first protein to Fig. 5). Pvfps are enriched with cysteine (Cys) and Tyr (Tyr)/ initiate interaction with the substrate, displacing interfacial water Dopa residues, with Pvfp-3a possessing 10 Cys residues, molecules before binding to a model metal (hydr)oxide surface accounting for 21 mol% of its amino-acid content, and Pvfp-5b (TiO anatase) via coordination complex interactions. Using 2 having 21 mol% Tyr side chains in its primary sequence (Fig. 2a). homology modelling and molecular dynamics simulations, we Amino-acid analysis determined ca. 11 mol% Dopa content in also highlight the role of adhesive proteins’ three-dimensional Pvfp-5b (10 residues), whereas a lower content was detected for (3D) structures in wet adhesion mechanisms. Notably, these Pvfp-3a (1 residue; B2 mol%) and Pvfp-6 (2 residues; B2 mol%) modelling studies indicate that Pvfp-5 adopts a mostly elongated, (Supplementary Fig. 6). Circular dichroism spectra for all Pvfps unordered structure, whereby all Dopa residues are positioned on exhibited predominantly random coil structures, with minima the protein surface to maximize surface interactions. However, between 200 and 205 nm (Fig. 2b). The positive ellipticity for Pvfp-5 is not fully disordered and structural stability is ensured Pvfp-5b between 225 and 240 nm corresponds to p–p* transition through the presence of b-strand domains and disulphide bridges. of the aromatic side chains, that is, Tyr/Dopa25,26. Dynamic Furthermore, Pvfp-5 contains multiple pairs of Dopa-lysine (Lys), light scattering (DLS) revealed a hydrodynamic diameter of which have recently been found to be critical in underwater 7.42±0.44, 9.50±0.26 and 24.13±0.84 nm for Pvfp-3a,-5b adhesion23. These findings have the potential to further refine and -6, respectively (Fig. 2c), indicating a different degree of current developments in biomimetic adhesives. They can also aggregation in solution (Supplementary Note 1). inspire environmentally friendly fouling-resistant technologies

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Structural modelling of Pvfps. To gain molecular-scale insights inevitably expected to be affected by micro-environmental into the role of Pvfps on green mussel adhesion, we modelled the changes, such as local pH and ionic strength. Given the estab- structure of Pvfp-3a, Pvfp-5b and Pvfp-6 by homology modelling lished role of the post-translational modification of Tyr to Dopa and molecular dynamics simulations (see Methods for details). in enhancing mussel adhesion, we modified all Tyr residues into Pvfp-5b and Pvfp-6 adopt similar folds consisting of an extended, Dopa (Pvfp-5b-Dopa). In comparison to Pvfp-5b simulations, the non-globular conformation. The structure of Pvfp-5b is predicted Tyr to Dopa modification does not reveal any significant struc- to contain a high percentage of b-strands (41% of the amino acids tural change (Supplementary Fig. 9a–c), and only patches of (aa)) forming anti-parallel b-sheets connected with loops (Fig. 3, negative potential appear on the Pvfp-5b-Dopa’s surface because Supplementary Figs 7 and 8). Pvfp-3a adopts a globular fold with of the presence of an extra hydroxyl group on the Tyr residues a high percentage of b-strands (38% aa) and an a-helical region (Fig. 4d). (aa 11–24). Pvfp-6 has a smaller percentage of b-strands (29% A detailed structural analysis of Pvfp-5b yields a conforma- aa), a small a-helix (aa 15–19) and a higher percentage of loops. tional fold in which the Tyr residues are located at the periphery The Cys residues in Pvfp-5b are all oriented such as to engage in of the molecule with their aromatic rings facing the solvent disulphide bridges. The a-helix in Pvfp-3a contains two cysteines, (Fig. 5a). We found that for all Tyr within all conformations, the which are also predicted to form disulphide bonds with a Cys solvent accessible surface area (SASA) is 88% larger than that of present in the C-terminal anti-parallel b-sheet region, thereby other amino acids (127.5±14.9 versus 67.6±6.0 Å2; Fig. 5c). A stabilizing the overall structure. Pvfp-5b shows a distribution similar trend was also observed for Pvfp-5b-Dopa, where the largely of positive charges over its surface, with high abundance at SASA was found to be further increased (144.3±13.6 versus the N-terminus region, and small patches of negative charges 67.9±5.9 Å2 for other amino acids; Fig. 5c). The probability of (Fig. 4b). In contrast, the surface electrostatic potentials for water molecules in the first solvation shell within 3.4 Å of any Pvfp-3a and Pvfp-6 display the presence of both negatively and atom of Tyr or Dopa showed an increase of 9% in the latter case positively charged regions (Fig. 4a,c). Such potentials are (Fig. 5b), clearly suggesting that the 8% increase in solvation of

a 1. Saline injection 2. Induced secretion 3. Plaque’s adhesion

Glass Foot Groove Adhesive KCI Induced plaque Injection byssus Byssal Pedal thread Nerve Glass Foot

bc d Glass Adhesive plaque Thread Induced byssus

efPvfp-3 Pvfp-5 Pvfp-6

Natural footprints 60

Pre-injection 8,000 9,000 10,000 11,000 12,000 50 g)

μ Pvfp–5, –3 and –6 10 s 100 40 %Int. 30 s 0 30 1 Min Pvfp–5 and –3

3 Min 20 Total protien ( Total

5 Min 11,000 11,500 12,000 10 Pvfp-5 10 Min 0 0 5 10 15 20 25 30 30 Min Time (min)

3,000 4,500 6,000 7,500 9,000 10,500 12,000 8,400 9,200 10,000 m/z (Da)

Figure 1 | Mussel adhesive proteins are secreted at discrete time intervals. (a) Schematic of the injection of the saline solution to induce the secretion of functional mussel’s byssus, as verified by the adhesion to glass via its plaque. (b) Mussel foot and (c) zoom in on the tip of the foot showing the freshly formed saline-induced byssal thread. (d) Adhesive plaque of the induced byssus adhering to a glass slide. The glued byssal thread was drawn out of the foot groove until the stem at the base of the foot. (e) MALDI-ToF MS spectra of native mussel footprint on glass (top spectrum), before saline injection (second from top) and at various time points (from 10 s to 30 min, bottom spectra) after injection. Coloured areas correspond to mass/charge (m/z) of Pvfp-3 (green), Pvfp-5 (red) and Pvfp-6 (blue). (f) Total proteins mass (±s.d.) secreted over time following KCl injection. Colour-coded areas denote the time intervals during which Pvfps are secreted (red: Pvfp-5; green: Pvfp-5 and -3; blue: Pvfp-5, -3 and -6). Dotted line is from a polynomial interpolation based on the average mass values. A total of 15 mussels were saline injected (see Supplementary Table 2 for full data).

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Pvfp-5b-Dopa arises chiefly from the increased solvation of Dopa. Another region of interest pertains to the stretching mode of We also computed the average distance of each amino acid to the OH groups (nOH) in the B3,800–2,800 cm À 1 region, which protein centre and found that the average distance of all Tyr primarily originates from water molecules involved in weak (18.6±2.3 Å) and Dopa (19.1±2.3 Å) is 16–19% higher than (liquid-like) and strong (surface-bound) hydrogen-bond for other amino acids in the respective structures (Fig. 5d). interactions at higher and lower wavenumbers, respectively30. Furthermore, the flexibility of the modelled Pvfp-5b, measured Negative bands progressively developed during the adsorption of À 1 as the N- to C-terminus distance over the last 60 ns of the Pvfp-5b on TiO2 (Fig. 6b) in the B3,800–3,300 cm and simulation varied from 72.07 Å (completely extended) to 49.43 Å B3,300–2,950 cm À 1 regions due to dislodgment of liquid-like (partially flexed; Supplementary Fig. 10 and Supplementary and surface-bound water molecules. Pvfp-5b thus exhibits the Movie 1). ability to displace interfacial water molecules from a wet hydrophilic surface (TiO2), subsequently enabling the formation of chemical bonds with the surface (inner-sphere complexes). Adsorption studies of Pvfps on TiO2. To experimentally assess the adsorption behaviour of Pvfps, we conducted quartz crystal a microbalance (QCM) and attenuated total reflection infrared Pvfp-3α : 47 Amino acids, 5.34 kDa pI=6.1 (ATR-IR) spectroscopy measurements. QCM plots (frequency Y= 2 (4%) changes Df versus time) for the adsorption of Pvfps on TiO were Dopa = 1 (2%) 2 C= 10 (21%) obtained by initially flowing buffer to obtain a stable baseline (equilibration step, see Methods). In three separate experiments, Pvfp-5β : 82 Amino acids, 9.45 kDa 100 ml of 0.1 mg ml À 1 Pvfps solutions were introduced for 2 min pI=9.2 (adsorption step) before washing with buffer to desorb weakly Y= 17 (21%) b Dopa = 9 (11%) bound Pvfps (desorption step). Pvfp-5 showed the greatest C= 12 (15%) adsorption (Dfads ¼ 12.8 Hz), followed by Pvfp-6 (Dfads ¼ 9.7 Hz), and finally by Pvfp-3a (Dfads ¼ 5.4 Hz; Fig. 6a). The frequency shift Pvfp-6 : 105 Amino acids, 11.39 kDa pI=6.6 normalized by the molecular weight, Dfads/molecular weight, is proportional to the number of protein molecules adsorbed on the Y= 8 (8%) Dopa = 2 (2%) QCM sensor. This value is highest for Pvfp-5b (1.32) and mini- C= 16 (15%) mum for Pvfp-6 (0.86), showing that the latter protein has the least affinity for the TiO2 surface. The greater adsorption of Pvfp-5b on b α TiO was associated with the fastest initial adsorption kinetics, as Pvfp-3 2 ) Pvfp-5β

–1 4 evident from the slopes at the beginning of the adsorption curve. Pvfp-6 After washing, Pvfp-6 showed the greatest desorption (Dfdes ¼ 1.8 ×dmol Hz), whereas Pvfp-5b and -3a desorbed in negligible amounts. 2 2 The fast and abundant desorption of Pvfp-6 may be attributed to a sizeable fraction of its adhesive Dopa residues being screened 0 (deg×cm

within Pvfp-6 aggregates, which were observed from DLS, thereby –3 reducing the availability of Dopa for surface binding. –2 ×10

In ATR-IR experiments, Pvfps were adsorbed from θ À 1 –4 0.1 mg ml solution in acetic acid and 0.25 M KNO3 on a B100-nm thick TiO nanoparticle film. Pvfp-5b adsorbed 2 –6 strongly on TiO2 with intense absorptions recorded already in Ellipticity, the first ATR-IR spectrum after 2 min (Fig. 6b). ATR-IR spectra 190 200 210 220 230 240 of adsorbed Pvfp-5b revealed absorptions associated with peptide Wavelength (nm) bonds, namely amide I, II and III at B1,640, 1,544 and À 1 27 1,242 cm , respectively . Other absorptions originating from c Pvfp-3α CH aliphatic vibrations28 were observed between B1,460 and Pvfp-5β 2 50 Pvfp-6 1,400 cm À 1. The broad band peaking at 1,091 cm À 1 is generally 24.13 nm associated with the bending mode (d) of CH groups and 8.56 nm d(CH) þ d(OH) combination mode29. In addition, the peak 40 7.42 nm at 1,520 cm À 1 due to aromatic residues (Tyr/Dopa) was also observed, with a prominent shoulder at 1,489 cm À 1 indicating 30 Dopa/Ti(IV) coordinative bond29. The amide I band is particularly sensitive to protein’s conformation and is routinely 20 27 used for determinations of their secondary structure . In the Intensity (%) initial ATR-IR spectrum, this band exhibited two peaks at 1,652 10 and 1,626 cm À 1, originating from random coils and b-sheets27, respectively, thus matching the modelled Pvfp-5b structure 0 (Fig. 3b and Supplementary Fig. 8). The evolution of the amide 0510 15 20 25 30 I band shape can be attributed to structural rearrangement of Hydrodynamic diameter, dH (nm) adsorbed Pvfp-5b. In fact, the two initial well-resolved peaks merged over the course of the adsorption, indicating the Figure 2 | Native Pvfps possess largely random coil structures and form emergence of a new contribution associated with b-sheets at aggregates in solution. (a) Amino-acid sequences22 of Pvfps isolated from 1,638 cm À 1 and the concurrent loss of random coils at mussel foot and corresponding molecular weight, pI and content of Tyr (Y in 1,655 cm À 1, as revealed by the difference spectrum between the blue), Dopa and Cys (C in red). (b) Circular dichroism spectra showing normalized initial and final ATR-IR spectra (Supplementary molar ellipticity (y) as a function of wavelength, and (c) DLS hydrodynamic À 1 Fig. 11). diameter (dH) distribution at 0.1 mg ml in acid saline buffer at pH 4.

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α1 abN-ter c

C-ter β1 N-ter β2 β2 β N-ter

Å 2 β 1 β3 β1 α1 β3 28.1 62.0 Å β4 62.8 Å β β3 7 β4 β5 β6 β5 β6 24.9 Å β α β8 7 Pvfp-3 β Pvfp-5 Pvfp-6 C-ter 24.0 Å C-ter 40.6 Å

Figure 3 | Molecular dynamics simulated structures of Pvfps display globular and elongated conformations. 3D homology models with average dimensions of (a) Pvfp-3a,(b) Pvfp-5b and (c) Pvfp-6 proteins with disulphide bonds shown in orange.

In adsorption phenomena, outer-sphere complexes with solvent a b separation, that is, electrostatic interactions between a solvated Pvfp-3α Pvfp-5β anion and a surface metal cation, can be the precursor of an 90° 90° inner-sphere complex, such as metal–ligand coordination bonds3. The presence of coordination bond between catechols (such as Dopa) and Ti(IV) is characterized by a doublet at ca. 1,490 and 180° 180° ca. 1,270 cm À 1 attributed to n(CC) of aromatic rings and n(CO) modes, respectively29,31. The negative second derivative of the ATR-IR spectra (Supplementary Fig. 12) confirmed the presence of a doublet at 1,482 and 1,270 cm À 1 in the initial spectra. The former peak gradually disappeared as the adsorption progresses c Pvfp-6 d Pvfp-5β−dopa and the TiO2 surface became saturated, indicating that Dopa side 90° 90° chains coordinated Ti(IV) leading to structural rearrangements and eventually to an enrichment of b-sheet in the adsorbed protein layer. Pvfp-3a and -6, in contrast, were not able to adsorb 180° 180° significantly on TiO2, perhaps because of their lower Dopa content as compared with Pvfp-5b. Removal of interfacial water is well recognized to be a major challenge in underwater adhesion1, and the presence of Dopa appears to be critical for enabling this behaviour. Figure 4 | Electrostatic potential maps show differences in surface charge distribution among Pvfps. Electrostatic maps for (a) Pvfp-3a,(b) Surface adhesion of adsorbed Pvfps layers. The adhesion Pvfp-5b,(c) Pvfp-6 and (d) Pvfp-5b-Dopa at 90° and 180° rotation around capability of Pvfps was assessed by surface force apparatus (SFA) their longitudinal axis. The surface potentials are represented from anionic experiments, using procedures established for Mytilus mussel (red) to cationic (blue) calculated at 0.25 M ionic concentration and 310 K. adhesive proteins16–18. When two mica surfaces were coated with layers of Pvfp-5b and brought into contact in acid saline buffer, corroborates the DLS result (Fig. 2c) that Pvfp-5b forms the initial force F measured on separating the surfaces was aggregates, most likely via Dopa–Dopa crosslinks. The largest adhesive (Fig. 7a). The maximum adhesion measured in four values of T were obtained on approaching the surfaces for the first independent experiments (with different pairs of mica surfaces) time at a given contact position, indicating that adsorbed À 1 32 was Fa/R ¼ 5.6 mN m , corresponding to a work of adhesion molecules and aggregates could be irreversibly compacted or À 2 W ¼ (2/3p)Fa/R ¼ 1.2 mJ m . Adhesion decreased with time as flattened on the substrate, perhaps due to the formation of the surfaces were repeatedly approached and retracted at a additional Dopa–Dopa covalent crosslinks or Dopa–mica contact position, and as different positions were tested (compare hydrogen bonds on compression. The force curve became first and third positions in Fig. 7a and Supplementary Fig. 13), approximately exponential at distances Do15 nm, with a decay most likely due to Dopa oxidation15. The average value (±sample length of about 2 nm up to the maximum load considered À 1 E À 1 s.d.) of the adhesive force was Fa/R ¼ 3.0±2.0 mN m . The F/R 20 mN m (Fig. 7a, inset). The same curve was obtained force was purely repulsive during surface approach, showing for multiple approaches at the same contact position. This that adhesion was generated by the bonds created by Pvfp-5b behaviour suggests that Pvfp-5b was adsorbed as a soft hydrated molecules during surface contact. The force consistently exceeded layer, as opposed to a compact ‘hard-wall’ coating, allowing water the detection threshold when the surface separation distance and protein molecules to move and rearrange as the compressive D became smaller than 2T ¼ 25–50 nm (Fig. 7a, inset). Since the force was increased. Debye length of the saline buffer was smaller than 1 nm (ref. 32), The force curves obtained after adsorbing Pvfp-3a on the mica such long repulsion range 2T can be attributed to the overlap surfaces of the SFA (Fig. 7b) showed a repulsion range between protein layers adsorbed on opposite surfaces, each 2TE50 nm and exponential regime at small distances, with d ¼ 3 having an approximate thickness T ¼ 12–25 nm. This value is nm and A ¼ 70 mN m À 1, similar to those obtained for Pvfp-5b. larger than the size of a Pvfp-5b molecule (Fig. 3b), and On the other hand, the adhesion force was significantly weaker:

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ab100 Tyr 100

Dopa 50 80 Pvfp-5β 480 520 560 600 60

40

Probability (%) 20 Pvfp-5β-Dopa

200 220 240 260 280 Number of water molecules in shell #1

c d 22 160 aa Pvfp-5β aa Pvfp-5β-Dopa aa Pvfp-5β aa Pvfp-5β-Dopa Tyr Pvfp-5β Dopa Pvfp-5β-Dopa Tyr Pvfp-5β Dopa Pvfp-5β-Dopa 140 20 120

per aa) 100 2 18 (Å) 80 avg 60 R 16

SASA ( Å 40 20 14 0

Figure 5 | The modelled structure of Pvfp-5b-Dopa is more spread out and hydrated than Pvfp-5b. (a) Representative structure with Tyr (cyan) and Dopa (magenta) at the periphery of the protein. (b) Probability of water molecules in the first hydration shell around all Tyr and Dopa residues. Inset shows the same probability for all amino acids combined in Pvfp-5b and Pvfp-5b-Dopa. (c) Average SASA and (d) average distance from

Tyr (dark cyan) or Dopa (dark magenta) to the protein centre (Ravg) and for all other amino acids for Pvfp-5b (light cyan) and Pvfp-5b-Dopa (light magenta). Average SASA and Ravg values were calculated for the equilibrium structure and error bars represent s.d.

a b 1,544 2 Buffer Pvfp Buffer Abs 0.0005 1,643 Abs 0.001 0 1,520 Surface-bound –2 water Liquid-like α –4 Pvfp-3 water 1,489 Time 1,412 1,091 –6 1,453 Pvfp-6 1,400 1,242 –8 Frequency (Hz) Frequency –10 Pvfp-5β

–12

–14 1,652 1,626 Time 0 2 46 4,000 3,600 3,200 1,700 1,600 1,500 1,400 1,300 1,200 1,100 1,000 Time (min) Wavenumber (cm–1)

Figure 6 | Pvfp-5b displaces surface-bound water from hydrated TiO2 and shows greater adsorption than other Pvfps. (a) QCM plots for the À 1 À 1 adsorption of Pvfp-3a,-5b and -6 on a TiO2-coated sensor. All proteins were adsorbed at a flow rate of 50 ml min from a 0.1 mg ml solution in acidic saline buffer. An initial equilibration period of B2 min was obtained by flowing the buffer, which was followed by the adsorption of the proteins (Pvfps), À 1 and finally by protein desorption under buffer flow. (b) ATR-IR spectra of adsorbed Pvfp-5b on TiO2 nanoparticle film from 0.1 mg ml solution recorded at À 1 À 1 B2-min intervals. Background was from acid saline buffer on TiO2-coated ZnSe prism. The B3,300–2,900 cm and B3,750–3,300 cm infrared regions correspond to surface-bound (yellow shade) and liquid-like (blue shade) water, respectively.

À 1 the maximum value measured in three independent experiments maximum adhesion force was Fa/R ¼ 1.7 mN m , smaller than À 1 was Fa/R ¼ 2.38 mN m and the average Fa/R ¼ 1.3±0.7 the values measured for either Pvfp-5b or Pvfp-3a alone (Fig. 7c). mN m À 1. Analysis of variance of the adhesion force data for Also the average value (0.8±0.8 mN m À 1) showed a significant Pvfp-3a (11 measurements) and Pvfp-5b (9 measurements) using decrease compared with Pvfp-5b (Po3% for two independent Welch’s t-test showed that the difference was statistically experiments and three different measurements). We hypothesize significant, with probability Po4% to be accidental (Fig. 7e). that the observed delay in secretion between Pvfp-3 and Pvfp-5 This result is in line with QCM and ATR-IR adsorption may help avoid such interference between Pvfp-3 and the measurements, showing that Pvfp-5b exhibits a more surface adhesive residues of Pvfp-5. When Pvfp-5b was first adsorbed adhesive character than Pvfp-3a. Since Pvfp-5b is the first and followed by Pvfp-3a, the maximum adhesion force decreased À 1 adhesive protein to be secreted after stimulating the mussel foot to Fa/R ¼ 2.6 mN m , close to the value measured for Pvfp-3a with saline injection (Fig. 1), it is interesting to study how Pvfp- alone (Fig. 7d). Therefore, Pvfp-3a appeared to almost completely 5b interacts with Pvfp-3a. When both Pvfp-3a and Pvfp-5b were screen and ‘replace’ the adhesive properties of the Pvfp-5b layer, simultaneously adsorbed from an equimolar mixed solution, the although statistical analysis indicates that the lower adhesion

6 NATURE COMMUNICATIONS | 6:8737 | DOI: 10.1038/ncomms9737 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9737 ARTICLE observed after adsorbing Pvfp-3a on Pvfp-5b compared with marine1 and freshwater mussels5,33, revealing that more intricate Pvfp-3a or Pvfp-5b alone may as well be due to random sequence features of mussel adhesive proteins are at play in experimental variations. These findings support the idea that enabling wet-resistant adhesion17–19. This study further unveils time-regulated secretion of Pvfps is designed at creating a critical time-dependant and molecular-scale mechanisms of functional multilayer at the interface between the adhesive plaque mussel adhesive proteins, using the Asian green mussel as a and the target surface for mussel attachment. In this multilayer model system. On triggering the process of byssus formation, configuration, Pvfp-5 is the main adhesive or primer in direct Pvfp-5 is consistently the first protein to be secreted, followed by contact with the surface, whereas Pvfp-3 provides the first Pvfp-3, and ultimately Pvfp-6. These findings are distinct from functional layer for the attachment of other proteins, such as those observed for Mytilus16, in which Dopa-rich mfp-3 is Pvfp-6. initially detected from the mussel foot, followed by mfp-6. A plausible explanation for the non-detection of the other plaque protein mfp-5 is perhaps related to the high laser power needed to Discussion desorb it from the plaque footprint by MALDI-ToF MS. Our understanding of mussel adhesion has broadened in recent It should be noted that upon saline injection, Pvfps are secreted years beyond the unambiguous role of Dopa chemistry in both over a longer timescale (ca. 15 min) compared with the natural

a Pvfp-5β b Pvfp-3α 10 100 10 100 d = 2 nm d = 3 nm –1 ) 8 10 A = 60 mN m–1 ) 8 A = 70 mN m

–1 10 –1 2T 6 1 6 1 2T (mN m 0.1 (mN m R

R 0.1

/ 4 / 4 F F 0.01 0.01 2 0204060 2 0204060

0 0

Force/radius, Force/radius, –2 / : 1st/3rd position –2 / : 1st/2nd position Fa/R –4 –4 0102030 40 50 60 0 102030405060 Distance, D (nm) Distance, D (nm)

cdPvfp-3α+Pvfp-5β Pvfp-3α on Pvfp-5β 10 10 ) 8 )8 Force/Radius, –1 –1 6 6 β (mN m Pvfp-5 only (mN m R

4 R / / : 1st/2nd position 4 / / Pvfp-3α added F F (1st/2nd position) 2 2

0 0

Force/radius, Force/radius, –2 –2 Force/radius, Force/radius,

–4 –4 0 102030405060 0 102030405060 Distance, D (nm) Distance, D (nm)

e 6 a 5 )

–1 Maximum Average 4

3 ab (mN m b R

/ 2 c F 1 0 Pvfp-3α on Pvfp-5β Pvfp-3α Pvfp-3α+Pvfp-5β Pvfp-5β

Figure 7 | Pvfp-5b has greater surface adhesion than Pvfp-3a and their mixture. (a,b) Normalized force F/R measured with the SFA as function of the surface separation distance D after adsorption of Pvfp-5b or Pvfp-3a on mica in saline buffer with pH 3. The insets show the range of repulsion 2T, force detection threshold (dotted line) and exponential curve F/R ¼ Ae À D/d.(c) Force measured after simultaneously adsorbing Pvfp-3a and Pvfp-5b from a mixed solution in acid saline buffer with pH 4. (d) Force measured after adsorption of Pvfp-5b on mica (black data points) and after adsorbing Pvfp-3a on Pvfp-5b (coloured points) from the same acid saline buffer with pH 4. Each symbol corresponds to one contact position and different colours indicate different surface approach/retraction cycles, with filled/open symbols indicating approach/retraction, respectively. The surfaces jumped out from adhesive contact at points indicated with arrows, where the adhesive force reached the minimum Fa/R.(e) Maximum and average adhesion force (F/R) performed in symmetric mode for Pvfp-5b, Pvfp-3a, Pvfp-3a þ Pvfp-5b equimolar mixture, and Pvfp-3a on Pvfp-5b. Results of Welch’s t-test comparisons are presented. Means that do not share a letter are significantly different.

NATURE COMMUNICATIONS | 6:8737 | DOI: 10.1038/ncomms9737 | www.nature.com/naturecommunications 7 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9737 secretion (ca. 1 min) (Supplementary Fig. 14 and Supplementary Supplementary Movie 1 and Supplementary Fig. 10) provide Movie 2). KCl solution injection into the mussel pedal ganglion structural plasticity to adapt the plaque protein to varying increases the concentration of K þ ions ([K þ ]) outside nerve cell conformational requirements, in particular for surface membranes, disrupting the K/Na steady state resting balance and, interactions39,42,43. 34,35 consequently, the resting potential (depolarization) . The Adsorption studies of native Pvfps conducted on TiO2, a model effect of the increase of [K þ ] on a cell membrane potential has surface frequently chosen to mimic metal oxide and mineral been shown for a frog muscle fibre: the membrane potential surfaces encountered by fouling organisms, reveal a clear ranking rapidly increased, and only a small repolarization occurred as of adsorption. Pvfp-5b showed the highest adsorption in QCM [K þ ] returned to normal, followed by rectification with Cl À assays, followed by Pvfp-3a and -6. ATR-IR spectroscopy ions36,37. Similarly, the saline injection depolarizes the mussel detected considerable spectral intensities from adsorbed Pvfp-5b pedal ganglion, turning on all foot processes. Unlike the well- binding to Ti atoms via coordination bonds. Pvfp-5b was able to coordinated on–off process of natural thread formation, the saline displace liquid-like and surface-bound water molecules from injection causes a sustained ‘on’ process that lingers until ion TiO2, thus overcoming repulsive hydration forces and enabling balance has been restored (J.H. Waite, personal communication). the formation of inner-sphere complexes. Pvfp-6 and -3a were It is important to notice that the saline-induced secretion does not detected, demonstrating their inability to strongly adsorb on nonetheless lead to the formation of a functional adhesive byssus wet hydrophilic surfaces. The characteristic doublet of Dopa/Ti (Fig. 1b–d). bidentate coordination gradually disappeared over time, as Three P. viridis adhesive proteins isolated from foot glands surface available sites became saturated. In agreement with the were re-dissolved in acidic solution at pH 4, which is consistent protein’s modelled structure, the initial ATR-IR spectrum of with the acidic pH imposed by marine mussels during plaque adsorbed Pvfp-5b showed a predominance of b-sheets and formation16,38. Pvfps display a predominantly random coil random coils, with the latter partially converting into b-sheets structure and form aggregates, with Pvfp-6 aggregates over time (Supplementary Fig. 11). Upon adsorption, the containing significantly more protein molecules than Pvfp-3a unordered conformation needs not to remain so, and an ordered and -5b aggregates (Supplementary Note 1). The 3D modelled structure can arise (that is, b-sheet) to stabilize the adsorbed structure of Pvfp-5b and Pvfp-6 adopt an extended, non-globular structure. The hydrophobic mfp-3S has been shown by Over- conformation with high aspect ratio, whereas Pvfp-3a is more hauser dynamic nuclear polarization relaxometry to be the only globular. In agreement with circular dichroism (Fig. 2b) and mfp able to evict hydration layers from a hydrophobic surface ATR-IR (Fig. 6b) spectroscopic analyses, all Pvfps are largely (polystyrene), although no mfp was able to displace water unstructured (that is, high content of random coils), with Pvfp-5b molecules from a hydrophilic surface (silica)44. In situ surface having the highest percentage of anti-parallel b-sheets (ca. 40%) dehydration studies of mussel adhesive proteins are challenging connected by loops (ca. 60%). Molecular dynamics simulations to carry out in laboratory conditions that mimic closely the revealed that all Tyr residues in Pvfp-5b are located on the natural environment. To this end, SFA and Overhauser dynamic protein surface and that they are 88% more exposed to the solvent nuclear polarization experiments pose difficulties. The former is than the other amino acids. When each Tyr in Pvfp-5b was performed under applied compressive forces and the latter converted into Dopa, the resulting molecular dynamics requires a surface to be spin labelled with nitroxide radicals. On simulations showed a further increase (9%) in the number of the other hand, ATR-IR spectroscopy used in this work is an water molecules solvating Dopa side chains compared with Tyr. in situ, label-free technique that allows unperturbed investigations Having 435% random coils, Pvfp-5b is considered highly of mussel adhesive proteins interacting with hydrated surfaces. unstructured39, which parallels the predominantly unordered SFA measurements complement the picture gained from QCM structure of Mytilus mfp-3 (refs 17,25,40) as measured by circular and ATR-IR adsorption studies, with Pvfp-5b exhibiting the dichroism. In previous molecular dynamics simulations, Qin and largest adhesive force when coated between mica surfaces, that is, Buehler41 assumed disordered structures for mfp-3 and -5 with an adhesion force of 3.0–5.5 mN m À 1, corresponding to a work exposed Tyr residues, although no homology modelling was of adhesion of 0.6–1.1 mJ m À 2. These values are in close carried out in this study. Using these assumptions, Tyr SASA agreement with those reported for the adhesion of Dopa-rich values in these proteins (78.8±15.0 and 109.3±12.7 Å2 per mfps adsorbed on mica under similar conditions of pH, salinity, amino acid for mfp-5 and -3, respectively) were significantly protein concentration and adsorption time1. In contrast, Pvfp-3a lower than those obtained for Pvfp-5b (127.5±14.9 Å2), which showed a weaker adhesion force (1.5–2.4 mN m À 1), in line with further increased when all Tyr are converted into Dopa previous SFA studies on mfps1. Notably, the co-adsorption of 2 (144.3±13.6 Å ). Furthermore, Ravg of Pvfp-5b comprising all Pvfp-5b and Pvfp-3a resulted in an adhesion force of Tyr and all Dopa residues is 18.6±2.3 and 19.1±2.3 Å, 1.7 mN m À 1, which is weaker than that of Pvfp-5b and Pvfp- respectively (16–19% higher than other residues), whereas those 3a alone. In other words, the co-secretion of these two proteins in for the assumed disordered structures of mfp-3 and -5 were more the early phase of mussel attachment would be detrimental for compact with Ravg of 11.0 and 9.3 Å, respectively (4–10% larger adhesion. Taken together, our adsorption and SFA measurements than other residues). Our homology modelling simulations thus thus quantitatively explain the stepwise secretion in mussel allow us to conclude that Tyr/Dopa residues of mussel adhesive adhesive proteins. Very recently, Maier et al.23 have revealed by proteins are considerably more exposed to the solvent than when SFA the synergistic interplay between Dopa and lysine (Lys) in assuming purely disordered structures17,25,41, with Pvfp-5b the bioadhesion of siderophores, a type of bacterial iron chelators. adopting a less-folded structure with a higher aspect ratio. Adjacent Dopa–Lys residues promote adhesion in seawater, Pvfp-5b conformation with all Tyr exposed to the solvent appears whereby Lys initially displaces hydrated surface cations and as a functional adaptation (i) to facilitate specific interactions with facilitates the subsequent Dopa binding to underlying substrates. tyrosinase, the enzyme involved in the posttranslational Interestingly, Pvfp-5b also comprises Lys in proximity to Dopa modification of Tyr into Dopa; and (ii) to maximize the density residues (three adjacent pairs and five pairs separated by one of Dopa at the protein surface. The modelled random coil amino acid; Fig. 2a and Supplementary Fig. 15). On the other N-terminal domain (amino acids 1–15), in particular, lacks a hand, Pvfp-3a and Pvfp-6 have no Lys and only one adjacent stable, specific 3D structure that along with the protein’s intrinsic Dopa–Lys pair, respectively. Together with the efficient water flexibility at its centre (extended to partially flexed transition; displacement capability and superior adsorption of Pvfp-5b, these

8 NATURE COMMUNICATIONS | 6:8737 | DOI: 10.1038/ncomms9737 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9737 ARTICLE sequence distinctions are in full agreement with the recent discovery that Dopa–Lys pairs are critical in enhancing Asian green mussel (Perna viridis) underwater adhesion. Considering the growing evidence that adhesive proteins in mussels and other fouling organisms are secreted as a complex 19,45,46 fluid, that is, coacervate , an interesting question will concern Groove Mussel foot the effect of homogenous solutions on adhesive proteins’ interactions and structures. Notwithstanding this complexity, a ~ 60 s clear model of wet bioadhesion in mussels of P. viridis emerges Pvfp-3 from our findings, whereby a Dopa-rich primer (Pvfp-5) is initially Pvfp-5 secreted because of its superior adsorptive and adhesive abilities, Pvfp-6 with a characteristic spread-out conformation to maximize interactions with foreign surfaces. The primer’s ability to displace surface-bound water from hydrophilic surfaces allows the formation of strong and durable bonds via its adhesive Dopa residues exposed on the protein surface, hence creating an environment conducive to the assembly of other plaque components (Fig. 8). The subsequent secretion of a Cys-rich Pvfp-3 protein (Pvfp-3) is likely to protect Dopa residues of Pvfp-5 from Pvfp-5 oxidation, through the formation of cysteinyl–Dopa bonds and disulphide bridges within the adhesive plaque, as observed for mfps. Given the high content of Cys within Pvfp-5b (15 mol%), another plausible scenario is that intramolecular disulphide bond formation could also protect Dopa residues from oxidation via redox chemistry. In other words, Pvfp-5b may be able to auto- protect itself against Dopa oxidation (and thereby minimizes loss of adhesion), a hypothesis that will be tested in future work. Time-regulated secretion of adhesive protiens Time-regulated secretion of adhesive

Methods Pvfp-5 Time-resolved protein secretion. The adhesive protein secretion in mussels of 0 s P. viridis was triggered by 1-ml injection of 0.56 M KCl, phosphate buffered at pH 7.2 into the mussel’s pedal nerve located at the base of the foot16,47,48. This process H O was demonstrated to mimic the mussel’s natural secretion, thus being virtually 2 48,49 indistinguishable . Proteins exuded were collected from the distal depression of Surface water displacement the mussel foot, the known location of plaque and adhesive protein secretion, by swabbing with a pipette tip, which was rinsed with 10 ml of 5% acetic acid. A total of n ¼ 15 mussels were considered. Proteins were collected before saline injection Figure 8 | Mussel wet adhesion is dictated by time-regulated secretion and at a specific time intervals after injection: 10 s, 30 s, 1, 3, 5, 10 and 30 min. of adhesive proteins. The Asian green mussel (on top) secretes adhesive The obtained fractions were analysed by MALDI-ToF MS by spotting 1.5 mlof protein solution sample on the MALDI plate and applying 1.5 ml sinapinic acid proteins (Pvfp-3, -5 and -6) from the groove located at the tip of its foot. (Sigma-Aldrich) as sample matrix. Quantification of the total protein concentration Pvfp-5 is the first protein to be secreted and is able to displace surface- collected at each time interval from the saline-induced mussel secretion was bound water molecules from wet surfaces. The modelled structure of determined spectrophotometrically by Bradford assay. A series of standard bovine Pvfp-5 indicates that all Dopa/Tyr residues are strategically located on the serum albumin (BSA) concentrations ranging from 0 to 2,000 mg ml À 1 (0, 25, 125, 250, 500, 750, 1,000, 1,500 and 2,000 mgmlÀ 1) were prepared by diluting an ampule periphery of the elongated protein to maximize the interactions with of albumin standard at 2 mg ml À 1 in 0.9% aqueous NaCl solution containing surfaces and other adhesive components. Furthermore, Pvfp-5 contains sodium azide (Thermo Scientific) with 5% acetic acid. The assay was performed by multiple pairs of Dopa–Lys located in close proximity from each other mixing 250 ml of the Coomassie reagent solution (Coomassie Bradford Protein Assay (Supplementary Fig. 15), which further contributes to surface water kit; Thermo Scientific), 5 ml of the BSA standard and each protein sample into a microplate (Nunclon 96 Flat Transparent, NUNCTM). The mixtures were allowed to displacement. Pvfp-3 is subsequently secreted in addition to Pvfp-5, incubate for 10 min at room temperature. The absorbance of each mixture was followed by the addition of Pvfp-6 in the last step of plaque deposition. measured at 595 nm with a plate reader (Infinite M200 Pro; TECAN). The protein concentration of the unknown samples was calculated from the equation obtained green, 4 M urea and 5% acetic acid) were prepared. Gels were pre-equilibrated with from the quadratic best-fit curve of the BSA concentration standard. the loading dye (10 ml per well) with a 5% acetic acid tank buffer at a constant current of 40 mA. Subsequently, a total volume of 10 ml of the prepared samples was loaded in each well. The gels were then run at a constant current of 40 mA. Isolation and purification of Pvfps Pvfps were extracted from the mussel’s foot . Two sets of gels were prepared for each sample to detect protein bands by staining organ of P. viridis mussels collected from the northern coast of Singapore (Johor with Coomassie Blue R-250 (Applichem; 0.125% Coomassie Blue R-250, 50% Strait 1.2643°N, 103.4226°E). Their phenolic glands were dissected and stored at methanol, 10% acetic acid), and redox-cycling proteins by NBT (Roche) (40 ml À 80 °C. Once frozen, the pigments on 50 glands were scraped off and the glands NBT in 50 ml of 2 M sodium glycinate at pH 10). These gels were then de-stained were homogenized in 150 ml of 5% acetic acid (Schedelco), containing protease with 7% acetic acid, 5% methanol and sodium borate solution. The molecular inhibitors, pepstatin and leupeptine (Thermo Scientific). The homogenate was weights of the purified proteins were identified by MALDI-ToF MS spectrometer centrifuged (S3033-High Speed Centrifuge J-26XP) at 20,000g at 4 °C for 40 min. (Axima-TOF2TM, Shimadzu Biotech). A 1:1 ratio of sinapinic acid matrix (Sigma- The supernatant was then collected for further purification by high-performance Aldrich; 20 mg ml À 1 and 1 ml) and the samples resuspended in 5% acetic acid (1 ml) liquid chromatography (HPLC) (Agilent 1,260 Infinity, USA). The protein was first were vortexed and loaded onto a MALDI plate. The samples were air dried and purified using a size-exclusion column (Agilent Bio SEC-5 columns, 150 Å) with a inserted into the instrument for analysis. 5% acetic acid mobile phase at 1 ml min À 1, which resulted in two major peaks after about 5 and 10 min detected at both 220 and 280 nm (Supplementary Fig. 3). The fractions of the peak B were pooled and ran through a reversed phase column Amino-acid analysis. The presence and amount of Dopa side chains in Pvfps were (Agilent ZORBAX 300SB-C8). The proteins were eluted with an aqueous determined by amino-acid analysis. Up to 30 ml of each protein were hydrolysed acetonitrile (Fulltime) gradient containing 0.1% trifluoroacetic acid (TFA) (Sigma- under vacuum at 110 °C with 150 ml of 6 M HCl (Fluka) and 10 ml phenol (Sigma) Aldrich) at 2 ml min À 1 (Supplementary Fig. 4). Acid urea polyacrylamide gel at 5% w/v for 8 h. The hydrolysates were flash evaporated using a speed vacuum electrophoresis with 10% acrylamide was performed to assess the purity of the (Scan Speed, USA), washing them twice with water and methanol. The proteins protein fractions. A 1:1 ratio of protein fraction and loading dye (0.15 mM methyl were then resuspended in 300 ml of sample dilution buffer (SYKAM, Germany).

NATURE COMMUNICATIONS | 6:8737 | DOI: 10.1038/ncomms9737 | www.nature.com/naturecommunications 9 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9737

The samples were centrifuged at 14 r.p.m. for 5 min. The supernatants were during the simulations for all the three models were stable as judged by the transferred into glass vials and loaded into an amino-acid analyser (SYKAM temporal evolution of r.m.s. deviation of Ca atoms. The simulations reached an Amino Acid Analyzer S433, Germany). Amino-acid standard (SKYAM, Germany) equilibrium state within B40 ns in all the simulations (Supplementary Fig. 7a), and and a Dopa standard (Sigma) at 1 mg ml À 1 dissolved in the sample dilution buffer therefore the analysis was carried out over the last 50 ns. Residue accessible surface were loaded. The amount of Dopa was then quantified by correlating the amino- area was calculated using Naccess V2.1.1 (ref. 62). The SASA, over the trajectory acid composition chromatogram of the Pvfp samples with that of the standards. for each amino acid was computed by assigning each atom an average radius of 2 Å and by summing up the total surface area. The trajectories were viewed in VMD63 and the images were created in VMD and PyMol64. The electrostatics calculations Circular dichroism. Pvfps were dissolved in 10 mM acetic acid at a concentration À 1 for each protein were carried out at 0.25 M ion concentration to replicate of 1 mg ml , and their secondary structure was determined by circular dichroism experimental conditions using the APBS plugin65 available in PyMol. using a Chriascan spectropolarimeter (Model 420, AVIV Biomedical Inc.). Mea- surements were conducted in triplicate at wavelengths ranging from 190 to 260 nm, with 1 nm step size and 1 nm bandwidth. The spectra were smoothed by the Quartz crystal microbalance. The 5-MHz piezoelectric quartz crystals coated Savitzky–Golay method with a polynomial order of 2. with TiO2 were purchased from Q-Sense, Sweden. The crystals were ozone treated for 10 min prior and after immersion in 2% SDS in purified water (Milli-Q, Dynamic light scattering. The hydrodynamic diameters of Pvfps were deter- Millipore) for 30 min. The cleaned surfaces were then washed thoroughly with mined by DLS. Proteins were solubilized in 10 mM acetic acid at 0.1 mg ml À 1 and purified water, dried with nitrogen gas and used immediately. Real-time QCM analysed with a 90Plus particle size analyser (Brookhaven Instruments) equipped measurements were performed using a Q-Sense E4 QCM (Q-Sense, Sweden) with a 658.0-nm monochromatic laser. To minimize the reflection effect, all instrument with four-channel IPC pump (Ismatec SA, Switzerland). Three cleaned crystals were mounted in the micro-fluidic chambers of the QCM. On attaining measurements were taken at a scattering angle of 90°. The number weighted À 1 histogram profiles of the proteins in solution were used for data analysis. stable resonant frequencies, 0.1 mg ml of each protein was injected in the three channels, respectively, at a constant flow rate of 50 mlminÀ 1. Pvfps were prepared in degassed 10 mM acetic acid at a concentration of 0.1 mg ml À 1. All experiments Homology modelling. There are no crystal structures available for the Pvfps, were repeated at least three times, with a s.d. of the resulting frequency change of so their 3D structures were generated using standard protein homology modelling o3% for the proteins used in the QCM adsorption experiments. methods. The full-length sequence of Pvfps-3a,-5b and -6 with 47, 82 and 105 residues, respectively20, was used as query to retrieve structural templates from the protein databank database using BLASTp50. This method searches for sequences Attenuated total reflection infrared spectroscopy. ATR-IR spectra were that are homologous to the query sequence. Only Pvfp-5b yielded two homologous recorded with a MIRacle accessory equipped with a three-reflection ZnSe prism structures with sequence identity 430%, namely DEL-1 EGF domain (PDB ID: (Pike Technologies) in an infrared spectrometer (Vertex 70, Bruker) equipped with 4D90, resolved at 2.60 Å (ref. 51)), a cell adhesion protein with 51% sequence a KBr beamsplitter and a liquid nitrogen-cooled mercury cadmium telluride identity, and Human NOTCH-1 EGFS domain (PDB ID: 2VJ3, resolved at 2.60 Å detector. The optical bench was continuously purged with nitrogen gas. The ZnSe B (ref. 52)), involved in the NOTCH signalling pathway, with 39% sequence identity. ATR prism was coated with a 100-nm thin film of TiO2 (anatase; o25 nm in Like Pvfp-5b, both proteins are disulphide rich. These two proteins were then used diameter) (Sigma-Aldrich) nanoparticles by drying 50 mlof10À 3 M sonicated 53 66–68 as templates to build a 3D model of Pvfp-5b using Modeller , a programme based aqueous TiO2 suspensions overnight in air . A volume of 40 ml of Pvfps at À 1 on comparative protein structure modelling by satisfaction of spatial restraints to 0.1 mg ml dissolved in 10 mM acetic acid and 0.25 M KNO3 was placed on the generate 10 models. Superimposition of all 10 models of Pvfp-5b generated using TiO2 nanoparticle film-coated ZnSe prism, and ATR-IR spectra were collected Modeller revealed conserved secondary structures with some differences in the from 128 co-added scans at 4 cm À 1 resolution with Opus software (version 6.5). conformations of the flexible loop regions. One structure was selected for our Background was from 10 mM acetic acid and 0.25 M KNO3 on the TiO2 film. The studies based on the lowest discrete optimized protein energy (DOPE) score54, spectra were corrected for the frequency dependence of the penetration depth with which ranged from À 5,580 to À 5,135. In the case of Pvfp-3a and Pvfp-6, no the ATR correction programme available in the Opus 6.5 software. template structure was identified with sequence identity 430%. To generate the 3D models of these two proteins, the automated I-TASSER pipeline55, which is based on multi-threading alignment and iterative template fragment assembly simulation, Surface forces apparatus. Adhesion and normal force measurements between was used. For consistency, a 3D model of Pvfp-5b was also generated using mica surfaces bearing adsorbed layers of Pvfps were obtained using a SFA (Mark III I-TASSER56 and was found to be similar to that generated using Modeller. The from Surforce LLC, USA). A detailed description of the technique can be found 69 Modeller-generated Pvfp-5b model with the lowest DOPE score, and the Pvfp-3a in Israelachvili and McGuiggan . Atomically smooth mica sheet were coated and Pvfp-6 models with low C-scores56 and high structural similarity to Pvfp-5b with a thin Ag layer on one side, glued on cylindrical glass supports with radius were chosen to be used as starting models for initiating molecular dynamics R ¼ 1.8–2.2 cm and mounted in the SFA in a crossed cylinder geometry. The radius simulations for additional refinement. Although the structures are composed of R and separation distance D of the surfaces at the single point of contact between many flexible regions (Supplementary Fig. 7b–d), visual inspection of the the cylinders were measured using an optical interferometry technique utilizing the constructive interference of light waves undergoing multiple reflections between trajectories revealed that the predicted disulphide bonds stabilize the structure 70 as well as maintain the tertiary shape of the protein. the partially reflective Ag coatings . Experiments with surfaces having different R were compared by considering the normalized force F(D)/R, which, according to Derjaguin relation, does not depend on R32. The distance D ¼ 0 was defined by the Molecular dynamics simulations. Simulations were performed using the set of wavelengths recorded at mica–mica contact in dry air (before adsorbing the Amber12 (ref. 57) ff99SB force field58. For simulations of Pvfp-5b modified with proteins). The error on F/R was smaller than 0.1 mN m À 1 (force detection Dopa, the partial charges and the force-field parameters were generated using the threshold in the insets of Fig. 7a,b) and the error on D about 0.5 nm. Pvfps were Antechamber module in AMBER. The Xleap module of Amber was used to prepare adsorbed on both mica surfaces from a 0.02 mg ml À 1 solution of protein in acid the system by adding hydrogen atoms. The structures were solvated using a box of saline buffer containing 10–100 mM acetic acid (with pHE4 and 3, respectively) 59 TIP3P water molecules . An octahedral box was constructed such that a minimum and 0.25 M KNO3 in purified water (Milli-Q grade from Millipore). Adsorption distance of 10 Å was ensured between any atom of the protein and the boundary of was stopped after 20 min by rinsing the surfaces with protein-free acidic solution. þ À the box. An appropriate number of Na and Cl counter ions were further added The homogeneity of the coated mica surfaces was assessed by atomic force to each system, to ensure net charge neutrality. All short-range, non-bonded van microscopy, with r.m.s. roughness of 8.91 and 1.89 Å for Pvfp-3a and Pvfp-5b, der Waals interactions were truncated at 9 Å and the particle mesh Ewald respectively (Supplementary Fig. 16). For each pair of mica surfaces we considered method60 was used to model the long-range electrostatic interactions. The SHAKE at least two contact positions. At each position, we measured at least two force algorithm61 was used to constrain bond vibrations involving hydrogen atoms. curves F versus D. Protein adsorption and force measurements were done at a fixed Solvent water molecules and counter ions were initially relaxed by energy temperature of 25±1 °C. minimization, keeping the protein atoms restrained. This was followed by relaxing the entire system using energy minimization to relieve any steric clashes. Each system was next subjected to molecular dynamics simulations using the following Atomic force microscopy. Atomic force microscopy images were acquired in the protocol: an equilibration phase where harmonic restraints were placed on the Ca a.c. mode in air on air-dried samples using an Asylum Cypher S (Asylum, Santa atom with a force constant of 25 kcal mol À 1 Å À 2 and the system gradually heated Barbara, USA). A Nanoworld NCSTR-50 cantilever with nominal resonant from 50 to 300 K for a period of 250 ps. This was followed by a gradual reduction of frequency of 160 kHz and force constant of 7.4 N m À 1 was used. Samples were the restraints over the next 250 ps until the restraints were reduced to 0; the system scanned at a rate of 1 Hz and 512 points per line resolution in a closed loop set-up. was allowed to evolve at 300 K over the next 2,000 ps without restraints. Finally, the equilibrated structures were subjected to three independent (different initial velocities) 100-ns molecular dynamics simulations (totalling 300 ns for each References system), and conformations were saved every 10 ps. Analysis of the simulations 1. Lee, B. P., Messersmith, P. B., Israelachvili, J. N. & Waite, J. H. Mussel inspired were carried out using the ptraj module. Before the analysis, the conformations wet adhesives and coatings. Annu. Rev. Mat. Res. 41, 99–132 (2011). generated in molecular dynamics simulations were centred and root mean square 2. Stewart, R. J., Ransom, T. C. & Hlady, V. Natural underwater adhesives. (r.m.s.) fitted to remove overall translation/rotations. The conformations sampled J. Polym. Sci. B Polym. Phys. 49, 757–771 (2011).

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Acknowledgements Competing financial interests: The authors declare no competing financial interests. We thank Prof. Herbert Waite for his teaching on the saline-induced mussel secretion and valuable discussions on the manuscript. We also thank Dr Garima Tiwari, Dr Dilraj Reprints and permission information is available online at http://npg.nature.com/ Lama and Mohan Pradhan for helpful discussions on the modelling and simulation reprintsandpermissions/ section. We are grateful to Batika Saxena for the artwork in Fig. 8. This research was How to cite this article: Petrone, L. et al. Mussel adhesion is dictated by time-regulated supported by the Maritime Port Authority (MPA) of Singapore through its Maritime secretion and molecular conformation of mussel adhesive proteins. Nat. Commun. Clean Energy Research Program (MCERP), and by the Singapore Maritime Institute 6:8737 doi: 10.1038/ncomms9737(2015). (grant no. SMI-2013-MA-03). S.A. is supported by a Singapore International Graduate Award (SINGA fellowship). We thank A*STAR Computing Resource Centre (A*CRC) This work is licensed under a Creative Commons Attribution 4.0 for computing facilities. International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise Author contributions in the credit line; if the material is not included under the Creative Commons license, L.P. and A.M. designed the overall study; B.Z designed SFA experiments and C.V. users will need to obtain permission from the license holder to reproduce the material. designed the molecular dynamics simulations; L.P. and C.N.S. extracted and purified To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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