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Oct 18, 2016 - Switch of Surface Adhesion to Cohesion by Dopa-Fe3+ Complexation, in Response to Microenvironment at the Mussel Plaque/Substrate...
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Switch of Surface Adhesion to Cohesion by Dopa-Fe3+ Complexation, in Response to Microenvironment at the Mussel Plaque/Substrate Interface Byeongseon Yang,† Chanoong Lim,‡ Dong Soo Hwang,‡ and Hyung Joon Cha*,† †

Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 37673, Republic of Korea School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang 37673, Republic of Korea

‡

S Supporting Information *

ABSTRACT: Although Dopa-Fe3+ complexation is known to play an important role in mussel adhesion for providing mechanical properties, its function at the plaque/substrate interface, where actual surface adhesion occurs, remains unknown, with regard to interfacial mussel adhesive proteins (MAPs) type 3 fast variant (fp-3F) and type 5 (fp-5). Here, we confirmed Dopa-Fe3+ complexation of interfacial MAPs and investigated the effects of Dopa-Fe3+ complexation regarding both surface adhesion and cohesion. The force measurements using surface forces apparatus (SFA) analysis showed that intrinsic strong surface adhesion at low pH, which is similar to the local acidified environment present during the secretion of adhesive proteins, vanishes by Dopa-Fe3+ complexation and alternatively, strong cohesion is generated in higher pH conditions similar to seawater. A high Dopa content increased the capacity for both surface adhesion and cohesion, but not at the same time. In contrast, a lack of Dopa resulted in both weak surface adhesion and cohesion without significant effects of Fe3+ complexation. Our findings shed light on how mussels regulate Dopa functionality at the plaque/substrate interface, in response to the microenvironment, and might provide new insight for the design of mussel-inspired biomaterials.

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Fe3+ complexation of fp-1 provides hardness, extensibility, and self-healing properties as the protective coating for all exposed surfaces of mussel byssus.18,22 It is a representative example of how protein−metal coordination is used to confer outstanding mechanical properties to biomaterials, rather than provide a physiological function. This sophisticated mechanical strategy has been applied to various protein-based biomaterials and synthetic polymers including hydrogels, nanofibers, and nanoparticles, as a type of material formulation and is frequently referred to as mussel-inspired.23−27 fp-3F and fp-5 have not been investigated as target proteins for studies on Dopa-Fe3+ complexation studies, despite exhibiting the highest Dopa content and the previous detection of Dopa-Fe3+ complex specific Raman resonance peaks across the byssal plaque, both at the plaque/substrate interface and the cuticle (Figure 1a).28 The MAP-Fe3+ complexation process has largely focused on fp-1, Dopa-conjugated synthetic polymers, and the mechanical properties that result from intermolecular bridging. The possibility of the Dopa-Fe3+ complexation of fp3F and fp-5 and the effects of complexation on mussel adhesion

INTRODUCTION Mussel adhesive proteins (MAPs) are widely known for their high 3,4-dihydroxyphenylalanine (Dopa) content. Dopa functions as the key molecule for mussel adhesion1−3 by mediating various interactions including bidentate hydrogen bonding,4−6 metal coordination,7,8 π−π/π-cation interaction,9,10 and oxidative cross-linking.11,12 In particular, a high Dopa content of 10−15 mol % was observed in MAP type 1 (fp-1), the main component of mussel byssus cuticle,13,14 and MAP type 3 fast variant (fp-3F) and type 5 (fp-5), surface adhesion components at the byssal plaque/substrate interface, have the highest Dopa contents of ca. 20 and 25 mol %, respectively (see Figure 1a, presented somewhat later in this paper).4,15,16 Dopa-Fe3+ complexation is one of the important interactions in Dopa mussel adhesion chemistry.14,17 Dopa can form stoichiometric mono-, bis-, and tris-complexes at different pH, and these complexes can be broken, as this interaction is reversible.18 In addition, the Dopa-Fe3+ complex is known to have a very high binding and stability constant and exhibits strong coordinate covalent bonds.19,20 Dopa-Fe3+ complexation has been observed at the mussel byssus cuticle.17,21 Cuticle protein fp-1 can form bis- and tris-complexes to Fe3+, recruit molecular bridging, and exhibit strong cohesion.14,17 The Dopa© 2016 American Chemical Society

Received: September 1, 2016 Revised: October 17, 2016 Published: October 18, 2016 7982

DOI: 10.1021/acs.chemmater.6b03676 Chem. Mater. 2016, 28, 7982−7989

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Chemistry of Materials

content (∼17 mol % for drfp-3F and ∼23 mol % for drfp-5), which are close to the Dopa content of natural fp-3F (∼20 mol %) and fp-5 (∼25 mol %) and facile acquisition, compared to the extremely difficult direct extraction from mussels (Figure 1b).31 We identified the Dopa-Fe3+ complexation of drfp-3F and drfp-5, and we investigated the effects of Dopa-Fe3+ complexation on underwater cohesion and surface adhesion by measuring cohesive and surface adhesive interactions using surface forces apparatus (SFA). Furthermore, we compared drfp-3F with Dopa-deficient recombinant fp-3F (rfp-3F) to suggest a possible mechanism for mussel byssal plaque formation.

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EXPERIMENTAL SECTION

Preparation of Materials. rfp-3F was produced in Escherichia coli and purified, as previously reported.38 The molecular mass of rfp-3F was confirmed to be 6652.9 Da by matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy (MALDI-TOF MS). rfp-5 was also produced in E. coli and purified, as previously reported.39 drfp3F was produced in E. coli via in vivo residue-specific Dopa incorporation and purified, as previously reported.31 In detail, the E. coli JW2581 tyrosine auxotroph was used as a host strain. The recombinant vector pQE-80L-fp-3h was transformed to E. coli. The cells were transferred to M9 minimal medium with 19 canonical amino acids, a limited amount of tyrosine (∼4 mg L−1), and ampicillin and were cultivated at 37 °C for ∼3−5 h to reach the stationary phase with an optical density at 600 nm (OD600) of ∼0.8. After confirmation of the stationary phase (depletion of tyrosine), Dopa was added at a concentration of 1 mM in the medium and the expression of drfp-3F was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG). The culture was further incubated at 37 °C and 220 rpm for 6 h. The molecular mass of drfp-3F was confirmed to be 6796.2 (9 Dopa residues) and 6812.4 Da (10 Dopa residues) by MALDI-TOF MS analysis. Amino acid composition analysis confirmed a Dopa incorporation yield of ∼94% and Dopa content of ∼17 mol %. drfp5 was also produced via in vivo residue-specific Dopa incorporation and purified in the same manner.31 Amino acid composition analysis confirmed a Dopa incorporation yield of ∼94% and a Dopa content of ∼23 mol %. Ultraviolet−Visible Light (UV-vis) Absorbance Spectrophotometry Analysis. To observe the spectral changes of mono-, bis-, and tris-Dopa-Fe3+ complexations, the absorbance of 2 g L−1 drfp-3F or drfp-5 in 10 mM acetic acid (pH ∼ 3) with 1 mM FeCl3 (Dopa:Fe3+ molar ratio of 3:1) was monitored on a UV-vis light microplate spectrophotometer (Bio-Rad, Hercules, CA, USA) from 350 nm to 850 nm at 5 nm increments. pH was adjusted by mixing the solution with same volume of buffer solution. The buffers included 0.2 M acetic acid (pH ∼3), 0.2 M sodium acetate buffer (pH 5.0), 0.2 M 3-(Nmorpholino)propane sulfonic acid (MOPS) buffer (pH 7.0), 0.2 M 2amino-2-hydroxymethylpropane-1,3-diol (Tris) buffer (pH 8.0), 0.2 M N-tris(hydroxymethyl)methyl-3-aminopropane sulfonic acid (TAPS) buffer (pH 9.0), and 0.2 M N-cyclohexyl-3-aminopropane sulfonic acid (CAPS) buffer (pH 11.0). For pH >11, 5% (w/v) NaOH was used. The spectral changes of rfp-3F and rfp-5 were also measured in the same manner. In this case, 2 g L−1 rfp-3F or rfp-5 solution in 10 mM acetic acid (pH ∼3) with 1 mM FeCl3 (tyrosine:Fe3+ molar ratio of 3:1) was monitored. UV-vis absorbance was analyzed, depending on the Dopa-Fe3+ pathway, as follows: (1) pH elevation after adding Fe3+ and (2) adding Fe3+ after pH elevation. drfp-3F (100 μL; 2 g L−1) was prepared. For (1), 1 μL of 50 mM FeCl3 was added and then 5 μL of 5% (w/v) NaOH was added. For (2), these steps proceeded in the reverse order: 5 μL of 5% (w/v) NaOH was added and then 1 μL of 50 mM FeCl3 was added. Finally, the UV-vis absorbance was measured from 350 nm to 850 nm in 5 nm increments. Resonance Raman Spectroscopy Analysis. The Raman spectrum was measured using a Raman microscope (WITec alpha300; Ulm, Germany). The diode-pumped, 633 nm laser

Figure 1. (a) Schematic illustration of mussel byssus. The figure indicates the location of each MAP from fp-1 to fp-6. The Dopa content is indicated by color. fp-3F and fp-5 are located at the plaquesubstrate interface mediating surface adhesion. Dopa-Fe3+ complexations are found all over the byssus from interface to cuticle. (b) Amino acid sequence information for drfp-3F and drfp-5; ∼94% of Dopa incorporation yield indicates that almost all tyrosine residues were converted to Dopa.

remain unknown. The presence of Dopa does not guarantee intermolecular bridging by complexation with Fe3+, and complexation is greatly affected by protein sequence and length.29,30 These bring three critical issues to debate: (1) the possibility of Dopa-Fe3+ complexation of Dopa-rich MAPs other than fp-1; (2) the effects on mussel adhesion, with respect to both surface adhesion and cohesion; and (3) the potential role of Dopa-Fe3+ complexation at the plaque/ substrate interface. Herein, we report Dopa-Fe3+ complexation at the plaque/ substrate interface and its effects on surface adhesion and cohesion. To address the above three issues, we used in vivo residue-specific Dopa-incorporated recombinant fp-3F (drfp3F) and fp-5 (drfp-5) as target plaque/substrate interfacial MAPs. These proteins were chosen because of their high Dopa 7983

DOI: 10.1021/acs.chemmater.6b03676 Chem. Mater. 2016, 28, 7982−7989

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Chemistry of Materials excitation was used in combination with a 20× microscope objective. The resonance Raman peak was detected with a charge-coupled device (CCD) camera (−60 °C). drfp-3F or drfp-5 solution (2 g L−1) with 1 mM FeCl3 (Dopa:Fe3+ molar ratio of 3:1) at pH ∼3, pH 8.0, and pH >11 was deposited on a glass slide, dried, and subjected to analysis. Raman spectroscopy analyses of rfp-3F and rfp-5 proceeded in the same manner. rfp-3F or rfp-5 solution (2 g L−1) with 1 mM FeCl3 (tyrosine:Fe3+ molar ratio of 3:1) at pH >11 was deposited on a glass slide, dried, and subjected to analysis. Surface Forces Apparatus Analysis. The Fe3+-mediated cohesion and surface adhesion forces were measured using a SFA (SFA 2000; SurForce LLC, USA) described in a previous report.40 For cohesion between symmetric drfp-3F films, a drfp-3F solution (0.1 g L−1 in 0.1 M acetic acid (pH ∼3)) was deposited onto freshly cleaved mica surfaces for 10 min, and the coated surfaces were rinsed with 0.1 M acetic acid solution. Then, a relatively high concentration of drfp-3F solution (0.5 g L−1 in 0.1 M acetic acid (pH ∼3)) was placed between the two mica surfaces to effectively prevent the bare mica surface from contacting the drfp-3F film and exclude asymmetric interactions. To test the effect of Dopa-Fe3+ complexation on cohesion, Fe3+complexed drfp-3F solution (0.5 g L−1 in 0.1 M acetic acid (pH ∼3) or 0.1 M Tris buffer (pH 8.0) without and with Fe3+(Dopa:Fe3+ molar ratio of 3:1)) was placed between two mica surfaces. For adhesion between the bare mica surface and an asymmetric drfp-3F film, drfp-3F solution (0.02 g L−1 in 0.1 M acetic acid (pH ∼3)) was deposited onto freshly cleaved mica surface for 10 min, and the surface was rinsed with 0.1 M acetic acid solution. To test the effect of Dopa-Fe3+ complexation on surface adhesion, a Fe3+ solution (0.5 μM of FeCl3 in 0.1 M acetic acid (pH ∼3)) was placed between two mica surfaces. Each interaction force and the separation distance were determined after 10 min of contact time. The measured force (F) is correlated to the energy per unit area (W) by the relation

W=

F 1.5πR

for a soft and deformable surface. The SFA analysis for the negative control rfp-3F also proceeded in the same manner. All SFA analyses were carried out with A compression time of 5 min and a contact time of 10 min, because it is regarded to be sufficient for forming a stable interaction between Dopa-Fe3+ complexation and physical entanglement of molecule chains.

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RESULTS AND DISCUSSION Identification of Dopa-Fe3+ Complexation of Plaque/ Substrate Interfacial MAPs. Dopa-Fe3+ complexation can easily be confirmed through observable color changes, as Dopa forms stoichiometric mono-, bis-, or tris-Dopa-Fe3+ complexes, depending on the pH.18,32,33 When FeCl3 was added to 0.5 g L−1 of drfp-3F solution up to a Dopa:Fe3+ molar ratio of 3:1, the transparent protein solution changed to purple/blue. Increasing the pH with NaOH resulted in the protein solution changing to pink/red, and decreasing the pH with acetic acid caused the protein solution to return to purple/blue, but not with full reversibility, because of Dopa oxidation (Figure 2a). UV-vis absorbance spectrophotometry analysis clearly confirmed the Dopa-Fe3+ complexation of drfp-3F solution with FeCl3 (Dopa:Fe3+ molar ratio of 3:1), exhibiting the characteristic shortening of absorption maxima wavelength with pH elevation as the dominant species changes from monocomplex (∼620 nm) to bis-complex (∼585 nm) and finally triscomplex (∼485 nm) (Figure 2b), a finding that has been shown in several reports.18,32,33 Dominant species were mono-/biscomplex at pH ∼3 with low intensity and bis-/tris-complex at pH 8.0. In contrast, rfp-3F did not show any absorbance as tyrosine cannot coordinate with Fe3+ (Figure S1a in the Supporting Information). Furthermore, the Dopa-Fe3+ com-

Figure 2. Dopa-Fe3+ complexation of drfp-3F and drfp-5. (a) Observation of the color changes of the protein solution and reversibility due to Dopa-Fe3+ complexation. (b) UV-vis absorbance of drfp-3F varies, depending on the pH. (c) Resonance Raman spectroscopy of drfp-3F with Dopa-Fe3+ complexation.

plexation of drfp-3F was confirmed with resonance Raman spectroscopy by detecting the seven characteristic resonance peaks resulting from Dopa-Fe3+ coordination, a finding that also has been shown in previous reports.18,28 In the case of pH ∼3, intensity was low but seven peaks were clearly detected while drfp-3F without Fe3+ did not exhibit any resonance peak, indicating that weak but certainly Dopa-Fe3+ complexation occurred, even at low pH. As expected, rfp-3F did not exhibit any resonance peak (Figure 2c). UV-vis absorbance spectrophotometry analysis and Raman spectroscopy were also performed for drfp-5 (see Figures S1b, S1c, and S1d in the Supporting Information). These results indicate that fp-3F and fp-5, which are small and specialized for surface adhesion, can also coordinate to Fe3+. These results also show that Dopa-Fe3+ 7984

DOI: 10.1021/acs.chemmater.6b03676 Chem. Mater. 2016, 28, 7982−7989

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mJ m−2) (Figure 3b). Because fp-3F is the smallest adhesive protein (6.8 kDa) adapted for surface adhesion, it is reasonable that no cohesion was observed. Under conditions that included Fe3+ in 0.1 M acetic acid (pH ∼3), drfp-3F showed a weak cohesive interaction (W = 0.507 ± 0.472 mJ m−2) (Figure 3b). This weak cohesion indicates that Fe3+ has an effect on the cohesive force between symmetric drfp-3F films. As described above, we also confirmed color changes in the protein solution at low pH and UV-vis absorbance near 600 nm as mono- and bis-Dopa-Fe3+ complexation become dominant. Intensity was very low, which means low pH is not a favorable condition for complexation. However, a small amount of Fe3+-mediated bridging cohesion may occur in the presence of a small proportion of bis-complexes, leading to weak cohesion at this low pH. 0.1 M Tris buffer (pH 8.0) with Fe3+ is the similar condition that mussel byssus meets when it is exposed to seawater, and the UV-vis absorbance analysis indicates that bis- and trisDopa-Fe3+ complexes are dominant under these conditions. As expected, SFA analysis measured a very strong cohesive interaction force under this condition (W = 2.126 ± 0.396 mJ m−2) (Figure 3b). During the move out, the two mica surfaces were not separated from each other at a certain point. Instead, analysis indicated that the distance between the two surfaces gradually increased during moving out primarily as a result of Fe3+-mediated bridging. This finding is thought to be the characteristic phenomenon of metal-mediated bridging and has been shown in several reports studying the cohesion of MAPs.14,28 The same phenomenon was also shown at pH 3 in the presence of Fe3+ (Figure 3b). This result was compared to the different behaviors of asymmetric adhesive interactions and non-metal-mediated symmetric cohesion in which detachment happens at a certain point.4,16,31 Under a condition of 0.1 M Tris buffer (pH 8.0) without Fe3+, drfp-3F showed a weak cohesive interaction force (W = 0.050 ± 0.015 mJ m−2) (Figure 3b). This result supports the theory that the strong cohesion of drfp-3F with Fe3+ at high pH originates from Dopa-Fe3+ complexation rather than O2/base-induced oxidation. In addition, film thickness was measured; while elevation of pH significantly increased film thickness, increased thickness did not always cause an increase in cohesion, showing no direct correlation between film thickness and cohesive interaction force (see Table S1 in the Supporting Information). As a result, we confirmed that the cohesive interaction of drfp-3F increased due to the addition of Fe3+ and elevation of pH. These findings indicated that the pH of seawater induces Dopa-Fe 3+ coordination, mediates intermolecular bridging, and result in strong cohesion between drfp-3F films. It is known that Dopa-Fe3+ coordination is dependent on sequence variation and consequently protein types and variants.29,30 Previous studies have shown that MAPs with subtle sequence differences could bind Fe3+ but have different effects on cohesion: while one MAP formed intermolecular complexes with strong cohesion, the other MAP formed intramolecular complexes without any effect on cohesion.29 Thus, we can conclude that fp-3F contains Dopa residues that can participate in intermolecular Fe3+ binding. fp-3F has a very high glycine content and is known to have unstructured extended coils.35 Thus, fp-3F may have relatively good flexibility, and its Dopa residues may have good accessibility to both the substrate for surface adhesion and other Dopa residues for cohesion. Thus, one can be expected that strong cohesion through Dopa-Fe3+ complexation is not only possible

complexation occurs at the plaque/substrate interface and may play certain roles in mussel adhesion. Analysis of Cohesion from the Dopa-Fe3+ Complexation of Plaque/Substrate Interfacial MAP. The cohesive force of the interaction between drfp-3F films was measured with SFA (Figure 3a). To exclude the asymmetric interaction

Figure 3. SFA analyses of the cohesive interactions of drfp-3F. (a) Schematic illustration of the SFA analysis used for measuring the cohesive interactions of drfp-3F. Both mica surfaces were coated with drfp-3F and a Dopa-Fe3+-complexed drfp-3F solution was deposited between two symmetric drfp-3F films. (b) SFA measurement was conducted under the following four different conditions: (1) in 0.1 M acetic acid (pH ∼3) without Fe3+, (2) in 0.1 M acetic acid (pH ∼3) with Fe3+ (Dopa:Fe3+ molar ratio of 3:1), (3) in 0.1 M Tris buffer (pH 8.0) without Fe3+, and (4) in 0.1 M Tris buffer (pH 8.0) with Fe3+ (Dopa:Fe3+ molar ratio of 3:1). The normalized forces (F/R) are shown on the left ordinate, whereas the corresponding interaction energies per unit area (defined as F/(1.5πR)) are on the right ordinate.

between insufficiently coated bare mica and the protein film, a high concentration of drfp-3F solution (0.5 g L−1) was placed between two protein-coated mica surfaces. SFA measurements were conducted under the following four different conditions: (1) 0.1 M acetic acid (pH ∼3), (2) 0.1 M acetic acid (pH ∼3) with Fe3+ (Dopa:Fe3+ molar ratio of 3:1), (3) 0.1 M Tris buffer (pH 8.0), and (4) 0.1 M Tris buffer (pH 8.0) with Fe3+ (Dopa:Fe3+ molar ratio of 3:1). The 0.1 M acetic acid condition (pH ∼3) was selected based on a recent report indicating an acidified local environment (pH ∼2−4) at the plaque/substrate interface during plaque formation.34 Because there is no clear information about the presence of Fe3+ in mussel-secreted interfacial MAPs, we tested both conditions with and without Fe3+ at pH ∼3. Condition (4) was included to investigate conditions similar to seawater that contain Fe3+ and exhibits a pH of ∼8.2 as the byssus is finally exposed to seawater. At pH ∼3 and in the absence of Fe3+, SFA measures the intrinsic cohesive interaction force between drfp-3F films. drfp3F showed almost no cohesive interaction (W = 0.041 ± 0.013 7985

DOI: 10.1021/acs.chemmater.6b03676 Chem. Mater. 2016, 28, 7982−7989

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adhesive force dramatically decreased, exhibiting very weak adhesion (W = 1.160 ± 0.339 mJ m−2) (Figure 4b). It was weaker surface adhesion than that of adhesive force at pH 8.0 without Fe3+ (W = 2.725 ± 0.219 mJ m−2) (Figure 4b), which means Dopa-Fe3+ complexation seems to decrease surface adhesion force additionally. Not only weaker electrostatic interaction, weaker salt displacement, and tendency to aggregate from reduced net positive charge at high pH but also the limited availability of Dopa for surface adhesion after bis- and tris-Dopa-Fe3+ complexation may cause a severe loss of drfp-3F surface adhesion. The addition of Fe3+ and an elevation of pH had a tendency to increase film thickness, but there was no direct correlation between film thickness and surface adhesive interaction force (see Table S1). As a result, drfp-3F showed very strong underwater surface adhesion at low pH, regardless of the presence of Fe3+ but significantly lost its surface adhesion under conditions where Dopa-Fe3+ complexation occurs, especially as bis- and tris-complexes. Our results showed that the addition of Fe3+ did not reduce the surface adhesive force at pH ∼3, indicating that the surface adhesion of Dopa residues apparently was not disrupted by Fe3+ mono binding. It is unclear whether the mono-Dopa-Fe3+ complex affects surface adhesion under force-free conditions. SFA analysis revealed no significant impact of mono Fe3+ binding on surface adhesion in the presence of compression. Dopa may retain its adhesiveness while binding Fe3+, or compression may break the mono-Dopa-Fe3+ coordination, recovering the adhesive functionality of Dopa as previously borate-chelated Dopa showed adhesion with compression.36 Fe3+ Effects on Dopa-Deficient Plaque/Substrate Interfacial MAPs. The cohesive and surface adhesive interaction forces of Dopa-deficient rfp-3F were also analyzed by SFA and compared to drfp-3F to investigate the role of Dopa in mussel adhesion related to Fe3+ complexation (Figure 5). The SFA measurement of cohesion was conducted under the same four conditions with Fe3+ (tyrosine:Fe3+ molar ratio of 3:1). At pH ∼3, without Fe3+, rfp-3F showed a weak cohesive interaction (W = 0.176 ± 0.177 mJ m−2). Under this condition, rfp-3F exhibited relatively better cohesion than drfp-3F, indicating that conversion from tyrosine to Dopa reduces cohesion. While drfp-3F showed a gradual increase in cohesion with the addition of Fe3+ and elevation of pH, rfp-3F did not show a pronounced increase in cohesion with the addition of Fe3+. Neither an elevation in pH (W = 0.037 ± 0.017 mJ m−2) nor the addition of Fe3+ (W = 0.268 ± 0.215 mJ m−2 at pH ∼3 and W = 0.094 ± 0.058 mJ m−2 at pH 8.0) could induce distinctive cohesion between symmetric rfp-3F films, because of the inability of tyrosine to interact with Fe3+ (see Figure 5a and Figure S2 in the Supporting Information). An SFA analysis of surface adhesion produced similar results. rfp-3F showed not only weak surface adhesive interaction force in the absence of Fe3+ (W = 2.720 ± 1.286 mJ m−2 at pH ∼3 and W = 1.951 ± 0.435 mJ m−2 at pH 8.0) but also no clear effect of Fe3+ (W = 2.393 ± 1.411 mJ m−2 at pH ∼3 and W = 3.802 ± 1.304 mJ m−2 at pH 8.0) (Figure 5b and Figure S3 in the Supporting Information). Weak rfp-3F adhesion indicates that the incorporation of Dopa is necessary to improve surface adhesion. We confirmed that (i) the surface adhesion of rfp-3F did not decrease at pH 8.0 with Fe3+, and (ii) it was better than the surface adhesion of drfp-3F under the same conditions. It seems that rfp-3F is least influenced by elevation of pH and the presence of Fe3+. As a result, the SFA analysis of Dopa-deficient rfp-3F showed little impact on cohesion and surface adhesion,

between fp-3Fs but also between other types of MAPs, thereby explaining the protein−protein interactions between MAPs in the byssal plaque. Analysis of Surface Adhesion from the Dopa-Fe3+ Complexation of Plaque/Substrate Interfacial MAPs. The effects of Dopa-Fe3+ complexation on surface adhesion were investigated by measuring the surface adhesive force between a bare mica surface and an asymmetric drfp-3F film using SFA (Figure 4a). The SFA measurement was conducted

Figure 4. SFA analyses of the surface adhesive interactions of drfp-3F. (a) Schematic illustration of the SFA analysis used for measuring the surface adhesive interactions of drfp-3F. Only one mica surface was coated with drfp-3F. (b) SFA measurement was conducted under the following four different conditions: (1) in 0.1 M acetic acid (pH ∼3) without Fe3+, (2) in 0.1 M acetic acid (pH ∼3) with 0.5 μM Fe3+, (3) in 0.1 M Tris buffer (pH 8.0) without Fe3+, and (4) in 0.1 M Tris buffer (pH 8.0) with 0.5 μM Fe3+. The normalized forces (F/R) are shown on the left ordinate, whereas the corresponding interaction energies per unit area (defined as F/(1.5πR)) are on the right ordinate.

under the same four conditions mentioned earlier, and the Fe3+ concentration was fixed to 0.5 μM. At pH ∼3, drfp-3F exhibited a very strong adhesive force on the mica surface (W = 14.728 ± 3.878 mJ m−2) (Figure 4b). Since fp-3F is known to be a surface adhesive protein located at the adhesive interface, these results indicated the strength of drfp-3F adherence to the wet surface in the presence of a high Dopa content. The addition of Fe3+ at pH ∼3 also resulted in a surface adhesive force (W = 15.267 ± 2.556 mJ m−2) similar to the value observed without Fe3+ (Figure 4b). It appears that Fe3+ mono binding did not interrupt the surface adhesion of Dopa residues. Surface adhesion was rather slightly enhanced. Partial combination of increased cohesion under this condition from imperfect monolayer formation of protein film and/or enhanced electrostatic interaction between negatively charged mica surface and additional positive charge from mono-Dopa-Fe3+ complexation is thought to be responsible for slightly enhanced surface adhesion. At pH 8.0 with Fe3+, however, the drfp-3F 7986

DOI: 10.1021/acs.chemmater.6b03676 Chem. Mater. 2016, 28, 7982−7989

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(mono-, bis-, or tris-) is dominant (Figure S5 in the Supporting Information). The Role of Dopa-Fe3+ Complexation at the Plaque/ Substrate Interface. The SFA measurements of the adhesive and cohesive interactions of rfp-3F and drfp-3F gave us very significant information about mussel adhesion. First, rfp-3F showed weak cohesion and weak surface adhesion at pH ∼3. While drfp-3F showed weaker cohesion than rfp-3F at the same low pH, drfp-3F showed very strong surface adhesion, indicating that surface adhesion to the substrate was significantly improved by a high content of Dopa, rather than tyrosine. A huge improvement in surface adhesion was obtained in exchange for cohesion reduction. On the other hand, drfp-3F showed strong cohesion by Dopa-Fe3+ complexation at pH 8.0 while rfp-3F did not. At this pH, drfp-3F lost its adhesion and exhibited lower surface adhesion than rfp-3F, indicating that cohesion was improved by sacrificing surface adhesion. Collectively, the high Dopa content of plaque interfacial MAPs causes stronger surface adhesion at low pH and stronger cohesion through Dopa-Fe3+ complexation at high pH. Mussels seem to ingeniously use this chemistry. Thus, a mussel adhesion mechanism at the plaque/substrate interface can be proposed (Figure 6). Mussels incorporate a high level of Dopa into MAPs for better adhesion and cohesion than tyrosine. Mussels construct a seawater-free, acidified, and isolated environment at the distal depression, resulting in a pH value

Figure 5. Comparative SFA analyses of rfp-3F and drfp-3F. (a) Cohesion energies (symmetric) and (b) surface adhesion energies (asymmetric) of Dopa-deficient rfp-3F (white bar) and fully Dopaincorporated drfp-3F (black bar) were measured by SFA under the following four different conditions: (1) in 0.1 M acetic acid (pH ∼3) without Fe3+, (2) in 0.1 M acetic acid (pH ∼3) with Fe3+, (3) in 0.1 M Tris buffer (pH 8.0) without Fe3+, and (4) in 0.1 M Tris buffer (pH 8.0) with Fe3+. The energies per unit area were defined as F/(1.5πR) and are shown on the left ordinate.

because of a lack of Fe3+ coordination and supports the theory that both the strong cohesion of drfp-3F with Fe3+ at high pH and strong surface adhesion of drfp-3F at low pH originate from Dopa residues. The Presence of Fe3+ in Mussel Secretion. The approximate pH and ionic strength present at the distal depression when the mussel foot secretes MAPs are known.12,34 However, there is no clear information regarding the salts present. The presence of Fe3+ in the vacuole prior to secretion of interfacial MAPs is also ambiguous. UV-vis absorbance revealed that increasing the pH after Fe3+ addition resulted in better absorbance and, therefore, better Dopa-Fe3+ complexation, than Fe3+ addition after pH elevation (Figure S4 in the Supporting Information). Thus, it is possible that MAPs contain Fe3+ ions in each vacuole before their secretion. The containment of Fe3+ did not reduce the surface adhesion of drfp-3F at low pH with compression, as mentioned above. Mussels are known to secrete MAPs with suction-based compression.37 The presence of Fe3+ in vacuoles needs to be further investigated and it will be interesting to determine the salt types and Fe3+ concentration in vacuoles, because the Fe3+ concentration determines the pH value where each complex

Figure 6. Schematic illustration of the proposed mechanism for mussel adhesion at the plaque/substrate interface. Mussel adhesion at the plaque/substrate interface involves two sequential steps. (Left) First, mussels generate a seawater-free, acidified (pH ∼2−4), and isolated environment at the distal depression into which MAPs are secreted. MAPs have a high Dopa content instead of tyrosine residues, which mediate only weak surface adhesion and cohesion. Because of their high Dopa content, MAPs show strong surface adhesion and begin to interact and adhere to the substrate surface. At this low pH, cohesion is very weak. (Right) After surface adhesion, the newly synthesized mussel byssus is exposed to seawater. The high pH and Fe3+ content of seawater encourage Dopa-Fe3+ complexation and induce strong cohesion between MAPs through intermolecular bridging. At this high pH, surface adhesion is very weak; however, the surface adhesion process has already been completed at this point and is not necessary. 7987

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Chemistry of Materials of ∼2−4, as previously reported, and secrete plaque/substrate interfacial MAPs such as fp-3F and fp-5.34 Cohesion is not the important factor under these conditions. The surface adhesion of Dopa is maximized and MAPs start to interact and adhere to the substrate surface. After surface adhesion, the newly synthesized byssus is exposed to seawater. The high pH and Fe3+ content of seawater result in Dopa-Fe3+ complexation and induce strong cohesion between MAPs. In conclusion, mussels use Dopa-Fe3+ complexation to switch the functionality of Dopa from surface adhesion to cohesion at the plaque/ substrate interface.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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CONCLUSION With regard to Dopa-Fe3+ complexation, many studies have focused on measuring cohesive interactions between proteins while neglecting surface adhesive force. This has occurred because the cuticle protein fp-1, which covers the entire exposed surface of byssus and is not used for surface adhesion, has been the main target protein for Dopa-Fe3+ complexation studies. However, fp-3F and fp-5 are adhesive proteins that have a high Dopa content, exhibit a very strong surface adhesive force, and are located at the plaque/substrate interface where surface adhesion actually occurs. It emphasizes the importance of understanding the adhesive properties of fp-3F and fp-5 related to Dopa-Fe3+ complexation. Here, the relationship between the surface adhesive properties of drfp-3F and DopaFe3+ complexation was analyzed and, to the best our knowledge, this is the first study to investigate surface adhesion as it relates to Dopa-Fe3+ complexation. A reduction in surface adhesion was observed in the conditions where bis- and trisDopa-Fe3+ complexation occurred. We also showed Dopa-Fe3+ complexation of drfp-3F and drfp-5, suggesting the possibility of Dopa-Fe3+ complexation at the plaque/substrate interface by natural fp-3F and fp-5. The cohesion of drfp-3F increases due to intermolecular bridging, thus illustrating the cohesion resulted from Dopa-Fe3+ complexation at the plaque/substrate interface. Thus, a possible mussel adhesion mechanism at the plaque/substrate interface can be proposed. Many mussel-inspired applications use Dopa-Fe3+ complexation.23−27 The use of Dopa-Fe3+ complexation seems to be a good strategy for improving mechanical properties, but this technique should be used carefully as a formulation for adhesives. The mussel adhesion mechanism is not simple as a high Dopa content increases the capacity for both surface adhesion and cohesion, but not at the same time. In that regard, the shift from surface adhesion to cohesion by Dopa-Fe3+ complexation is very similar to the regulation of Dopa oxidation by oxidants with the exception of reversibility.11,38 We believe that this study has identified sophisticated mussel adhesion mechanism capable of responding to the Fe3+ and pH of microenvironments. The optimal balance between the surface adhesion and cohesion generated by Dopa-Fe3+ complexation must be considered in designing mussel-mimicking biomaterials with targeted applications.

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cohesion of rfp-3F; UV-vis absorbance studies dependent on the complexation pathways and Dopa:Fe3+ ratio (PDF)

ACKNOWLEDGMENTS We acknowledge the financial support provided by the Marine Biotechnology program (Marine BioMaterials Research Center) funded by the Ministry of Oceans and Fisheries, Korea. We thank Dr. K. Cho (POSECH) and Mr. M. S. Yoo (POSECH) for assistance with the resonance Raman spectroscopy analyses.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.6b03676. UV-vis absorbance and Raman studies of rfp-3F, rfp-5, and drfp-5; SFA analysis of the surface adhesion and 7988

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