Self-Assembled Nanofibers for Strong Underwater Adhesion: The

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Self-Assembled Nanofibers for Strong Underwater Adhesion: the Trick of Barnacles Chao Liang, Zonghuang Ye, Bin Xue, Ling Zeng, Wenjian Wu, Chao Zhong, Yi Cao, Biru Hu, and Phillip B. Messersmith ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b04752 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018

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ACS Applied Materials & Interfaces

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Self-Assembled Nanofibers for Strong Underwater

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Adhesion: the Trick of Barnacles

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Chao Liang,†,‡ Zonghuang Ye,† Bin Xue,‡ Ling Zeng,† Wenjian Wu,† Chao Zhong,§ Yi Cao,*,‡

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Biru Hu,*,† and Phillip B Messersmith∥,⊥

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†

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Technology, Changsha 410073, P. R. China, ‡Collaborative Innovation Center of Advanced

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Microstructures, National Laboratory of Solid State Microstructures, Department of Physics,

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Nanjing University, Nanjing 210093, P. R. China, §School of Physical Science and Technology,

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ShanghaiTech University, Shanghai 201210, P. R. China, ∥Departments of Materials Science

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and Engineering and Bioengineering, University of California, Berkeley, Berkeley, CA 94720,

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United States, and ⊥Materials Science Division, Lawrence Berkeley National Laboratory,

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Berkeley, CA 94720, United States

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KEYWORDS: barnacle; biophysics; force spectroscopy; proteins; self-assembly; underwater

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adhesion

Department of Chemistry and Biology, College of Science, National University of Defense

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ABSTRACT: Developing adhesives that can function underwater remains a major challenge for

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bioengineering; yet, many marine creatures, exemplified as mussels and barnacles, have evolved

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their unique proteinaceous adhesives for strong wet adhesion. The mechanisms underlying the

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strong adhesion of these natural adhesive proteins provide rich information for biomimetic

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efforts. Here, combining atomic force microscopy (AFM) imaging and force spectroscopy, we

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examine the effects of pH on the self-assembly and adhesive properties of cp19k, a key barnacle

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underwater adhesive protein. For the first time, we confirm that the bacterial recombinant

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Balanus albicostatus cp19k (rBalcp19k), which contains no 3,4-dihydroxyphenylalanine

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(DOPA) or any other amino acids with post-translational modifications, can self-assemble into

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aggregated nanofibers at acidic pHs. Under moderately acidic conditions, the adhesion strength

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of unassembled monomeric rBalcp19k on mica is only slightly lower than that of a commercially

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available mussel adhesive protein mixture, yet the adhesion ability of rBalcp19k monomers

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decreases significantly at increased pH. In contrast, upon pre-assembled at acidic and low-

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salinity conditions, rBalcp19k nanofibers keep stable in basic and high-salinity seawater and

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display much stronger adhesion and thus show resistance to its adverse impacts. Besides, we find

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that the adhesion ability of Balcp19k is not impaired when it is combined with an N-terminal

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Thioredoxin (Trx) tag, yet whether the self-assembly property will be disrupted or not is not

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determined. Collectively, the self-assembly enhanced adhesion presents a previously unexplored

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mechanism for the strong wet adhesion of barnacle cement proteins and may inspire the design

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of barnacle-inspired adhesive materials.

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INTRODUCTION

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Inhabited in the wave-swept intertidal regions, barnacles (Figure 1a) and mussels (Figure 1b)

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have evolved the unique ability to firmly anchor themselves to almost any underwater hard

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substrates, by secreting and curing of multi-protein adhesives.1, 2 The barnacle is notorious as a


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dominant marine fouling organism all around the world.3 Its underwater adhesion is so robust

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that external forces usually break barnacle peripheral shells before destroying the adhesive joint.

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As a result, the basal plates of barnacles are still tenaciously glued to the substrate while their

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parietal plates and bodies have been removed (Figure 1c). From barnacle underwater cement, a

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number of protein components have been isolated and characterized so far.4-9 Among them, a 19-

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kDa cement protein (cp19k) is suggested to locate at the interface between external substrata and

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bulk cement and play the critical role of binding to various underwater substrates (e.g., ship hulls,

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rocks and the shells of other marine creatures) with different surface properties.7,

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understanding how cp19k adaptively and strongly binds to different substrates is invaluable for

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the design and engineering of strong synthetic underwater adhesives, as well as the prevention of

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barnacle fouling on target surfaces. Unfortunately, the underlying mechanism remains largely

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unknown presently.

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Thus,

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Cp19k is possibly functionally analogous to the mussel foot protein-5 and -3 (mfp-5 and -3)

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located at the interface between mussel adhesive plaque and foreign substrate (Figure 1d).11, 12

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The abundant DOPA (3,4-dihydroxyphenylalanine), modified from Tyr, in mfp-5 and -3 plays a

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key role in their substrate binding by mediating a variety of different interfacial interactions.13-19

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Moreover, the neighboring positively charged amino acids were shown to be able to collaborate

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with DOPA to enhance the surface binding ability of mfp-5 and -3.20-22 In contrast, cp19k does

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not have DOPA or any other post-translationally modified amino acids. It is characterized by the

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biased amino acid composition that six amino acids (Ser, Thr, Gly, Ala, Val and Lys) altogether

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account for approximately 70% of the total residues.7, 23-25 Furthermore, the arrangement of those

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amino acids exhibits the characteristic of block copolymers. As illustrated in Figure 1d, the

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primary structure of Balanus albicostatus cp19k (Balcp19k) consists of two alternating blocks.


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One is rich in hydrophobic and charged amino acids, such as Val and Lys, whereas the other

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contains a high abundance of Ser, Thr, Gly and Ala.1, 9 Therefore, the substrate binding of cp19k

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likely relies on the unique composition and arrangement of common amino acids, rather than

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post-translational modifications.1, 10 However, the structure-function relations of cp19k have not

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been fully discussed so far. Owing to the versatility of DOPA, mussel-inspired DOPA-tethered

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adhesive materials have made huge progress in recent years; however, the uncontrollable redox

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chemistry of DOPA makes it difficult to achieve optimal adhesion in certain circumstances.26-28

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In this regard, cp19k may provide novel paradigms to design more promising barnacle-inspired

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adhesives.

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Figure 1. Underwater adhesion of barnacles and mussels. (a) A cluster of barnacles attaches

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themselves on a mussel shell by secreting and curing of proteinaceous barnacle cement. (b) A

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mussel clings itself to rocks by a bundle of byssi. Each byssus is distally anchored to the


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substrate by the expanded adhesive plaque, which is made up of a variety of mussel foot

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proteins. (c) Residual barnacle basal plates on a polymer substrate. The basal plates of barnacles

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are still firmly glued to the substrate by barnacle cement whereas their peripheral shells and

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bodies have been removed. All the scale bars represent 10 mm. (d) Three adhesive proteins

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playing the key role of substrate binding in barnacles and mussels. Balcp19k, Balanus

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albicostatus cp19k, its primary structure shows the characteristic of block copolymers. Balcp19k

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contains two alternating blocks: one is rich in hydrophobic and charged amino acids (orange

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background), whereas the other is dominated by Ser, Thr, Gly and Ala (blue background). Mefp-

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5 and Mefp-3, foot protein-5 and foot protein-3 in Mytilus edulis. They have abundant DOPA

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and most DOPA residues are flanked by positively charged amino acids. Dopa (Y) and Lys (K)

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were highlighted in the three proteins in red and blue, respectively.

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In this study, employing circular dichroism (CD), atomic force microscopy (AFM) imaging

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and force spectroscopy, we tracked the structural, morphological and functional changes of the

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bacterial recombinant Balanus albicostatus cp19k (rBalcp19k) at different pHs, aiming to reveal

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its possible pH-dependent self-assembly and adhesive properties.

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EXPERIMENTAL SECTION

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Construction of the recombinant plasmid. Plasmid construction was performed using In-

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Fusion HD cloning kit (Clontech, CA) according to the user manual. First, the coding sequence

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of Balcp19k cDNA was amplified from our previously constructed plasmid via a pair of specific

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primers: 5’-GAC GAC AAG GCC ATG GTG CCC CCC ACC GTG CGA C-3’ (Balcp19k IF-F)

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and 5’-GCT CGA ATT CGG ATC CTC AGA GTC CCT TCA ACT CGA G-3’ (Balcp19k IF-

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R).29 Second, by recognizing the underlined sequences, the In-Fusion HD enzyme catalyzed fast


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and efficient ligation between gel-purified PCR products and linearized pET-32a(+) plasmids.

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The constructed recombinant plasmid was confirmed by sequencing.

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Protein expression and purification. The new fusion protein Trx-Balcp19k was expressed in

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Escherichia coli host strain BL21 (DE3) using the same protocol as previously reported,29 and

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purified on AKTA pure 25 (GE Healthcare) with pre-packed columns. Crude Trx-Balcp19k was

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purified by affinity chromatography (AC) with HisPrep FF 16/10 column from the lysates (in

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Buffer A: 20 mM Na2HPO4, 500 mM NaCl, 20 mM imidazole, pH 7.4). Non-target proteins

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were rinsed off by 10% Buffer B (20 mM Na2HPO4, 500 mM NaCl, 500 mM imidazole, pH 7.4)

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while target proteins were eluted by 40% Buffer B, at a constant flow rate of 5 ml/min. The

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eluted crude Trx-Balcp19k was dialyzed against Buffer C (50 mM Tris-HCl, pH 7.4) over night

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and then loaded onto HiPrep SP HP 16/10 column for ion-exchange chromatography (IC). A

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gradient procedure of 50% Buffer D (50 mM Tris-HCl, 1 M NaCl, pH 7.4), 40 min was applied

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to purify Trx-Balcp19k at a constant rate of 2.5 ml/min, and target Trx-Balcp19k was eluted at

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about 20% Buffer D. Finally, the collected Trx-Balcp19k fraction was concentrated and changed

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to the enzymatic digestion buffer (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl2, pH 7.4) through

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ultracentrifugation.

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To remove the fused tags, purified Trx-Balcp19k was then digested by recombinant bovine

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Enterokinase (Neptunus Interlong, Shenzhen) at following optimized conditions: 50~100 µg of

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Trx-Balcp19k per international unit (IU) of Enterokinase, 16°C for 45 min. To isolate tag-free

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rBalcp19k, digestion products (~1 ml) were loaded onto HiTrap SP HP 1 ml column for IC. A

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gradient procedure of 50% Buffer D, 50 min was applied at a constant flow rate of 1 ml/min, and

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target rBalcp19k was eluted at ~25% Buffer D. Using IC only, we failed to isolate high-purity

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rBalcp19k, therefore, reversed-phase high-performance liquid chromatography (RP-HPLC) was


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adopted for further polishing of rBalcp19k. RP-HPLC was conducted on a Waters HPLC system

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with an analytical Zorbax SB C3 column (300 Å, 4.6×250 mm) (Agilent) using Solution A (95%

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Milli-Q water, 5% Acetonitrile, 0.1% TFA) and Solution B (5% Milli-Q water, 95% Acetonitrile,

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0.085% TFA). A gradient procedure of 25%-65% Solution B, 50 min was applied at a constant

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velocity of 1 ml/min, and target protein was eluted at around 35% Solution B. Due to the low

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aromatic amino acid content of rBalcp19k, which results in extremely low optical density at 280

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nm (OD280), we monitored both OD280 and OD215 in all chromatography. Polished rBalcp19k

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was lyophilized for long-term storage. Using the same RP-HPLC procedure, we also polished

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Trx-Balcp19k and Trx tag, which were served as controls in following experiments. The final

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products were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-

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PAGE), protein N-terminal sequencing and matrix-assisted laser desorption/ionization-time of

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flight (MALDI-TOF) mass spectroscopy.

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Preparation and modification of colloidal probes. A colloidal probe was made by gluing a

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glass sphere with a diameter of ~20 µm to the triangular cantilever of Bruker MLCT AFM

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probes (nominated spring constant of ~0.07 N/m) using Devcon 5 Minute Epoxy (No. 14250,

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Danvers, MA). The accurate diameter of the attached glass sphere was measured by FEI Quanta

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200 scanning electron microscope (SEM) after the adhesion test. Purified rBalcp19k (or controls)

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was covalently immobilized to the colloidal probe according to the method described by Guo et

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al. with minor modifications.30 Briefly, prior to use, the colloidal probe was washed by ethanol

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bath for 1 h, rinsed by Milli-Q water and dried in air, followed by 20-min cleaning at room

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temperature in an UV Ozone cleaner (NovaScan) to introduce more surface hydroxyl groups.

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Then, the colloidal probe was silanized via reacting with 2% (3-Aminopropyl)triethoxysilane

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(APTES) in toluene for 2 h. The adsorbed excess APTES was rinsed by toluene and the probe


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was cured at 80°C for 20 min. Next, the glutaraldehyde linker was grafted to the silanized probe

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by soaking it in 2.5% glutaraldehyde in 10 mM PBS (pH 7.4) for 2 h. The weakly adsorbed

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glutaraldehyde was removed by rinsing with excess PBS and the linker-modified probe was then

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reacted with 0.5 mg/ml rBalcp19k (or controls) in 10 mM PBS (pH 7.4) for 1 h, to immobilize

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the protein onto the colloidal probe. The modified colloidal probe was kept in 10 mM PBS (pH

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7.4) at 4°C and used within 1 day.

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AFM based force spectroscopy. To characterize the microscale adhesion ability of

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rBalcp19k, AFM based force spectroscopy was carried out on JPK NanoWizard II using protein

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modified colloidal probes at ambient temperature (22 ± 2°C). Aiming to reveal the impacts of pH

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on rBalcp19k adhesion, mica was adopted as a model surface due to its inert surface chemistry

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and easy preparation. Before collecting force curves, the spring constant of the probe was

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calibrated by thermal noise method. During experiment, the probe was controlled to approach the

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mica substrate at a constant speed of 1 µm/s till the load reached 3 nN. Then, the probe was kept

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in contact with the surface at the constant load for 2 sec, and retracted at a pulling rate of 1 µm/s

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to a Z length of 1 µm. By repeating the process, a total number of 4×100 force curves were

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collected from 4 different sites on the mica substrate. To examine the effect of pH on the

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adhesion ability of rBalcp19k, force spectroscopy was successively conducted using the same

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probe in 4 different buffers: (1) 10 mM sodium acetate buffer (pH 3.6) and 150 mM NaCl, (2) 10

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mM sodium acetate buffer (pH 5.0) and 150 mM NaCl, (3) 10 mM sodium phosphate buffer (pH

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8.0) and 150 mM NaCl, and (4) 10 mM sodium carbonate buffer (pH 9.9) and 150 mM NaCl. In

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order to compare the adhesion ability of rBalcp19k with other well-known adhesive and non-

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adhesive proteins, the microscale adhesion forces of commercial Cell-Tak cell and tissue

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adhesive (Corning, MA), which was composed of natural DOPA-containing mussel adhesive


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proteins extracted from Mytilus edulis, Trx-Balcp19k and Trx tag were also examined under the

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same experimental conditions.

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The collected force profiles were analyzed by JPK Data Processing software and Igor Pro. In a

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typical retracting force curve, the jump-out peak at zero probe-substrate separation indicates the

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adhesion (Fad) between protein and surface. For each group of force data, a histogram was drawn

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to show the distribution of Fad, and Gaussian fit was made to obtain the most probable adhesion

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force that was further divided by the radius (R) of the glass sphere to obtain the normalized

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adhesion force (Fad/R). Based on the Johnson-Kendall-Roberts (JKR) theory, the relationship

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between surface adhesion force (Fad) and surface adhesion energy (Ead) of sphere/planar surface

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contact is Fad =3πREad, if one of the solid bodies is elastic and deformable.31 In our experimental

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setup, the protein layers (with a thickness ranges from a few nanometers to a dozen nanometers)

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coated glass sphere can be considered as an elastic and deformable solid body while the mica

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substrate is rigid, and thus, the equation was applied to calculate Ead.

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CD spectroscopy. CD spectra were recorded on J-815 CD spectrometer (Jasco) using a quartz

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cuvette with a light path of 1 mm. First, rBalcp19k was dissolved in the aforementioned 4 buffers

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at ~2.0 mg/ml and incubated at 4°C. After 0 day (without incubation), 1 day, 3 and 5 days, the

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stock solutions of rBalcp19k were 10-fold diluted with 10 mM buffers (without NaCl) having

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corresponding pH values, and their CD spectra from 190 nm to 260 nm were then collected. CD

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spectra of the buffers were also recorded and subtracted from those of rBalcp19k solutions. The

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background-subtracted CD spectra were smoothed and their unit was converted from θ (mdeg) to

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molar ellipticity (deg cm2 dmol-1). The contents of different secondary structures, including α

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helix, β sheet, turns and random coils, of rBalcp19k incubated in different buffers for different

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time were calculated from CD spectra using online DichroWeb.32, 33


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AFM imaging. In a previous study, we found that a bacterial recombinant Cys-substituted

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Balcp19k mutant was able to self-assemble into amyloid-like fibers at seawater analogous

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conditions.34 Therefore, in the present study, we first examined the self-assembly properties of

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rBalcp19k and its mutant (See reference 34 for the detailed procedure of producing the mutant)

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in simulated seawater conditions (pH 8.0, I=600 mM). Second, the nanoscale morphologies of

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rBalcp19k and its mutant at different pHs were examined by AFM imaging to compare the

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possible pH-dependent self-assembly properties. As for sample preparation, rBalcp19k and its

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mutant were firstly dissolved in different buffers at ~2.0 mg/ml and incubated at 4°C for 3 days.

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Then, 10 µl of the protein solution were aliquoted onto a newly peeled mica surface and allowed

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to adsorb for 30 min. Excess protein solutions were carefully rinsed with Milli-Q water. After

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dried in air, the samples were scanned by intermittent contact mode in air using Nanosensors

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PPP-NCHR probes.

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Thioflavin T (ThT) binding assay. The rBalcp19k (2.5 mg/ml) was dissolved in 10 mM

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sodium acetate buffer (pH 5.0) containing 150 mM NaCl and incubated at 4°C for self-assembly.

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In a period of 19 days, every other day, 10 µl of rBalcp19k stock solution were sampled and

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mixed with 5 µl of 1 mM ThT and 235 µl of 10 mM Tris-HCl (pH 8.0), to make the final

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concentrations of rBalcp19k and ThT were 0.2 mg/ml and 20 µM, respectively. The mixture was

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set at room temperature for 5 min, and its fluorescence emission spectra were subsequently

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recorded from 460 nm to 600 nm on a Jasco FP-6500 spectrofluorometer with 450 nm excitation.

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Emission spectra of the buffers were subtracted from those of the protein solutions, and the

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fluorescence emission at 482 nm of the background-subtracted spectra was recorded and plotted

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as a function of time.


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Hen egg-white lysozyme (HEWL) was adopted as a positive control to show the typical

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growth kinetics of amyloid fibrils.35, 36 HEWL (Amresco, OH, USA) was dissolved in 10 mM

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HCl (pH 2.0) supplemented with 0.05% NaN3 at 20 mg/ml and incubated at 65°C for amyloid

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fibril formation. At different days, 2.5 µl of HEWL stock solution were sampled and mixed with

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10 µl of 1 mM ThT and 487.5 µl of 10 mM Tris-HCl (pH 8.0), to ensure the final concentrations

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of HEWL and ThT were 0.2 mg/ml and 20 µM, respectively. Using the same parameters, the

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fluorescence emission of the mixture at 482 nm was examined in a period of 19 days too. The

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characteristic growth kinetics of HEWL amyloid fibrils was obtained by plotting the 482-nm

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fluorescence emission as a function of days.

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Evaluation of the stability of rBalcp19k nanofibers in seawater. AFM imaging was used to

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evaluate the stability of pre-assembled rBalcp19k nanofibers in seawater. First, pre-assembled

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rBalcp19k nanofibers were prepared by incubating rBalcp19k solution (1.3 mg/ml in a pH 5.0,

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I=150 mM buffer) at 4°C for 30 days. After one-month self-assembly, the presence of rBalcp19k

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nanofibers was confirmed by AFM imaging. Second, the pH and ionic strength of rBalcp19k

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solution were adjusted to 8.0 and 550 mM, respectively, to mimic seawater conditions. It was

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incubated at 4°C for another month, and at different days (1 day, 3 and 30 days), the morphology

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of pre-assembled rBalcp19k nanofibers were examined to see whether rBalcp19k nanofibers will

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disassemble or not.

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Examination of the adhesion ability of rBalcp19k nanofibers. The adhesion ability of self-

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assembled rBalcp19k nanofibers was characterized using the method reported by Zhong and

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coworkers.37 Firstly, 10 µl of pre-assembled rBalcp19k nanofibers (~0.5 mg/ml) were adsorbed

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on the mica substrate, and then force profiles were collected on protein coated surface with new

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colloidal probes made from Nanosensors SSS-SEIHR-50 probes that have an average spring


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constant of ~21 N/m. Prior to use, the colloidal probes were cleaned using the same protocol as

2

described earlier. To collect force profiles, the probe was controlled by an AFM to repetitively

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approach and retract from protein coated mica substrate, with a load of 500 nN, a contact time of

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2 sec and a pulling rate of 1 µm/s. In each buffer, force curves were collected from at least 6

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different locations on the substrate, and at each position a minimum of 20 force profiles were

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recorded. The same probe and protein coated substrate were used to acquire force curves in 4

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different buffers to examine the effects of pH and ionic strength on the adhesion ability of

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rBalcp19k nanofibers. The above experiment was repeated twice using different probes and

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protein adsorbed mica substrates. Data analysis was performed in JPK data processing software

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to obtain the average adhesion force (Fad), which was further divided by R (probe radius) and

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3πR to get normalized adhesion force and adhesion energy, respectively.

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RESULTS AND DISCUSSION

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Due to the great challenge of isolating native Balcp19k from barnacle cement, we expressed

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and purified rBalcp19k from E. coli. Despite that some researchers have already produced

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bacterial recombinant Balcp19k,29,

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sequence and conformation of rBalcp19k next to its native version. Our obtained rBalcp19k

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shows high purity as seen from the SDS-PAGE results in Figure 2. It has an apparent molecular

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weight (Mw) of ~23 kDa estimated from SDS-PAGE while an exact Mw of 17.5 kDa judged

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from MALDI-TOF (Figure S1). The rBalcp19k introduces only two additional amino acids (Ala-

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Met) at the N-termini of natural Balcp19k, and has one intramolecular disulfide bond formed

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between the two conserved Cys residues (See Table S1 for the sequences of rBalcp19k and

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controls). Notably, the Balcp19k used in this study (Table S1) shows two amino acid distinctions

34, 38

in this study, we made every endeavor to make the


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(L69F, L106I) compared to the one submitted to the GenBank database (Figure 1d, Accession

2

No. 242295.1).

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Figure 2. SDS-PAGE results of rBalcp19k and controls. Lane L: pre-stained protein ladder; lane

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1: Trx-Balcp19k; lane 2: rBalcp19k; lane 3: Trx. The different migration rate of rBalcp19k in the

6

presence (+DTT) and absence (-DTT) of dithiothreitol (DTT) indicates that the two Cys residues

7

in rBalcp19k have formed an intramolecular disulfide bond.

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As depicted in Figure 3a, in order to investigate the adhesion ability of rBalcp19k, it was

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covalently immobilized to the colloidal probe via silane chemistry and a glutaraldehyde linker to

10

perform AFM based force spectroscopy. Figure 3b shows a representative SEM photograph of a

11

modified colloidal probe, with which thousands of force profiles were sequentially recorded in 4


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Page 14 of 32

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buffers with the same ionic strength (150 mM) and different pH on a mica substrate. Some

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representative force profiles of rBalcp19k and controls were shown in Figure 3c and Figure S2,

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respectively, which were normalized by the radius (R) of the attached SiO2 sphere. In retracting

4

force curves, the jump-out peak at zero probe-substrate separation represents the adhesive

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interactions between proteins and mica. Figure 3d summarizes the normalized adhesion force

6

and adhesion energy of rBalcp19k and controls (see Table S2 for detailed results). It can be seen

7

that all proteins showed strong interfacial adhesion at acidic conditions (pH 3.6 and 5.0) while

8

weak adhesive interactions at basic environments (pH 8.0 and 9.9), suggesting that at low-pH

9

conditions, the electrostatic attraction between positively charged proteins (See Table S1 for

10

their isoelectric points) and negatively charged mica surface played a major role. Besides, thanks

11

to the relatively strong bidentate hydrogen bonding of DOPA on mica,39 Cell-Tak, a commercial

12

DOPA-containing mussel adhesive protein mixture, showed high adhesion force of 1.54 ± 0.59

13

mN m-1 at pH 3.6 and 0.82 ± 0.19 mN m-1 at pH 5.0. At basic pHs, the adhesion force of Cell-

14

Tak decreased significantly, because of the reduced net positive charges of mussel adhesive

15

proteins and self-oxidation of DOPA.39, 40 The adhesion force of rBalcp19k on mica at pH 3.6

16

(0.88 ± 0.24 mN m-1) and pH 5.0 (0.65 ± 0.17 mN m-1), mainly resulted from the attractive

17

electrostatic interactions, was smaller than that of Cell-Tak. Due to reduced positive surface

18

charges, under basic conditions, rBalcp19k showed decreased but slightly higher adhesion force

19

than Cell-Tak. Trx-Balcp19k, the hybrid protein of thioredoxin (Trx) and Balcp19k (Table S1),

20

had comparable adhesion force with rBalcp19k under all conditions, probably implying that the

21

adhesive function of Balcp19k can be retained or even enhanced when Balcp19k is combined

22

with suitable peptide tags.29 This finding suggests that cp19k can be potentially used as an


14

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1

adhesive tag to construct various protein-based biomaterials.41 As expected, the negative control

2

Trx (Table S1) showed the lowest adhesion force in all buffers.

3 4

Figure 3. Characterization of the microscale adhesion ability of rBalcp19k using AFM based

5

colloidal probe technique. (a) Schematic of the microscale adhesion force test. The rBalcp19k

6

was firstly conjugated to the colloidal probe via APTES and glutaraldehyde. Subsequently, the

7

modified probe was controlled by an AFM to approach and retract from the mica substrate

8

repetitively. During approaching and retracting, force profiles were recorded by plotting the

9

vertical deflection of the probe (Force) as a function of probe-substrate separation. (b) A SEM

10

photograph of the modified colloidal probe. The probe was made by attaching a SiO2 sphere with

11

a diameter of 20~30 µm to the end of an AFM cantilever. (c) Representative force profiles of

12

rBalcp19k under different conditions. The jump-out peak at zero probe-substrate separation of

13

the retracting force curve indicates the adhesion force (Fad) of rBalcp19k on mica, which is

14

normalized by the radius (R) of the SiO2 sphere. The corresponding adhesion energy is acquired

15

by dividing Fad with 3πR. (d) Normalized adhesion force and adhesion energy of rBalcp19k and


15


Page 16 of 32

1

controls under various conditions. The results were averaged from at least three independent

2

trials of 4×100 force curves.

3

Subsequently, we detected the secondary structure and nanoscale morphology of rBalcp19k at

4

the aforementioned four different conditions, to investigate whether cp19k has pH-triggered

5

structural and morphological transitions. After incubated under different conditions for different

6

time, CD spectra of rBalcp19k were collected and the results were shown in Figure 4a and

7

Figure S3. It can be seen clearly that the secondary structure of rBalcp19k showed time- and pH-

8

dependent transition behavior. Without incubation (t=0 day), rBalcp19k had similar CD spectra

9

(a strong negative peak at ~200 nm and a weak positive peak at ~215 nm) in different buffers.

10

Whereas after incubated at 4°C (t=1 day, 3 and 5 days), it exhibited significantly different CD

11

spectra under acidic conditions: the negative peak at ~200 nm vanished while a new negative

12

peak at ~222 nm and a new positive peak at ~200 nm emerged. Using DichroWeb (on-line

13

analysis for protein CD spectra), we found that the secondary structure of rBalcp19k (t=0 day)

14

was dominated by random coils and β sheets and only had extremely low content of α-helix

15

(Table S3), which is consistent with the results of Wang and coworkers.38 When incubation time

16

was increased, under acidic conditions, the α-helix content of rBalcp19k increased a lot while the

17

β-sheet percentage remained unchanged, whereas at basic pHs, the secondary structure of

18

rBalcp19k did not show any obvious changes within 5 days (Table S3). In line with the time- and

19

pH-dependent structural transition of rBalcp19k, using AFM imaging, we found that rBalcp19k

20

also showed pH-dependent self-assembly property: at pH 3.6 and 5.0, rBalcp19k self-assembled

21

into short nanofibers whose average diameter was around 1 nm, whereas at pH 8.0 and 9.9, no

22

nanofibers and only small rBalcp19k particles were observed (Figure 4b, c, e, f). In order to

23

understand the self-assembling dynamics of rBalcp19k nanofibers, we examined their


16

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1

morphologies at different time points after initiating self-assembly and the results were shown in

2

Figure S4. After self-assembly at pH 5.0 and 4°C for 24 hours, we observed isolated single

3

rBalcp19k nanofibers and small aggregates of rBalcp19k nanofibers. Three days later, almost no

4

single rBalcp19k nanofibers could be detected and most rBalcp19k nanofibers entangled to form

5

larger aggregates. When self-assembly time was further increased, rBalcp19k started to

6

precipitate probably due to the high extent of entanglement and aggregation of self-assembled

7

nanofibers. After one month, the vast majority of rBalcp19k formed visible insoluble pellets in

8

the buffer (pH 5.0, I=150 mM), but rBalcp19k fiber aggregates with relatively low degree of

9

aggregation could still be detected.

10

Given that the pH-dependent self-assembly of rBalcp19k resembles the formation of amyloid-

11

like structures of many unrelated proteins under low-pH conditions, next, we carried out ThT

12

binding assay to examine whether rBalcp19k nanofibers were typical amyloid-like fibrils. ThT, a

13

fluorescent dye, is extensively used to identify amyloid fibers and once it binds to amyloid

14

fibers, its fluorescence intensity at 482 nm (excitation at 450 nm) will be significantly enhanced.

15

42, 43

16

increase at all during a self-assembly period of 19 days, despite that massive rBalcp19k

17

nanofibers have been detected after 3 days (Figure S5). The result was in accord with our

18

discovery that the α-helix content of rBalcp19k increased greatly while the β-sheet (structural

19

basis of amyloid-like fibers) content did not show any obvious changes during self-assembly

20

(Table S3). In contrast, the positive control, HEWL, showed typical lag-growth-plateau kinetics

21

of amyloid fibers assembly.44 Based on these results, we speculated that the rBalcp19k

22

nanofibers formed under acidic conditions might be not typical amyloid-like fibers. However,

From Figure 4d, it can be seen that the ThT fluorescence intensity of rBalcp19k did not


17


Page 18 of 32

1

considering the non-specificity of ThT,42, 43 more rigorous structural characterizations may be

2

necessary to conclude whether they are amyloid-like fibrils or not.

3 4

Figure 4. Structural and morphological characterizations of rBalcp19k under various conditions.

5

(a) CD spectra of rBalcp19k in different buffers. D0: t=0 day (without incubation); D3: t=3 days.

6

(b, c, e, f) AFM height images showing distinct morphologies of rBalcp19k under different

7

conditions. At pH 3.6 and 5.0, rBalcp19k self-assembled into short nanofibers with an average

8

diameter of around 1 nm, while at pH 8.0 and 9.9, only small rBalcp19k particles were detected.

9

(d) ThT binding assay of rBalcp19k at different days during its self-assembly. The fluorescence

10

intensity at 482 nm did not increase during rBalcp19k self-assembly, indicating that rBalcp19k

11

nanofibers might be not amyloid-like fibers. The positive control, HEWL, showed typical growth

12

kinetics of amyloid fibers.

13

The self-assembly property of barnacle cement proteins makes them different from well-

14

studied mussel and sandcastle worm adhesive proteins, since no studies have reported their self-


18

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1

assembly ability up to now, to the best of our knowledge. Nakano et al. firstly confirmed that

2

peptides designed from cp20k and cp52k could self-assemble into ordered nanostructures.45, 46

3

Christopher et al. also discovered that recombinant Mrcp20k, cp20k in Megabalanus rosa, was

4

able to self-assemble into nanofibrils on calcite surface.47 Recently, Liu et al. investigated the

5

self-assembly property of cp19k using a Cys-substituted rBalcp19k mutant.34 Combining ThT

6

binding assay with transmission electron microscope (TEM) observation, they discovered that

7

the rBalcp19k mutant could self-assemble into amyloid-like fibers in simulated seawater

8

conditions. At acidic and low-salinity conditions, they failed to detect amyloidogenic self-

9

assembly via ThT staining.34 However, these results were obtained with a cp19k mutant, whose

10

self-assembly property may differ from its wild-type version. Moreover, they did not examine its

11

morphology at acidic and low-salinity conditions to see whether the protein could form non-

12

amyloid fibers. With this in mind, in the present study, we systematically compared the self-

13

assembly properties of rBalcp19k and its mutant under a variety of conditions. We found that

14

both rBalcp19k and its mutant formed nanofibers in simulated seawater conditions (Figure S6).

15

Interestingly, we observed that the rBalcp19k mutant self-assembled into nanofibers at all pHs

16

(Figure S6). The different self-assembly properties of rBalcp19k and its mutant under basic

17

conditions probably indicates that the intramolecular disulfide bond may have an effect on the

18

self-assembly of Balcp19k, however, the speculation needs further confirmation.

19

What are the advantages of using nanofibers for underwater adhesion? We propose that it can

20

prevent cp19k from being over diluted after secretion and enhance its interfacial adhesion in

21

seawater. To verify this hypothesis, we subsequently examined the morphology and adhesion

22

ability of pre-assembled rBalcp19k nanofibers in simulated seawater conditions. Firstly, we

23

prepared pre-assembled rBalcp19k nanofibers by incubating rBalcp19k dissolved in a pH 5.0,


19


Page 20 of 32

1

I=150 mM buffer at 4°C for 30 days. After one month, nanoscale rBalcp19k fiber aggregates

2

were successfully detected (Figure 5a). Secondly, the pH and ionic strength of the solution were

3

adjusted to 8.0 and 550 mM, respectively, to mimic seawater conditions. The sample was

4

incubated at 4°C and at different days the morphology of pre-assembled rBalcp19k nanofibers

5

was examined. As shown in Figure 5b-d, pre-assembled rBalcp19k nanofibers were stable in

6

seawater and did not disassemble within 30 days. To evaluate the microscale adhesion ability of

7

rBalcp19k nanofibers, they were physically adsorbed on the mica substrate rather than

8

conjugated to the probe, because most rBalcp19k nanofibers entangled to form insoluble pellets

9

in the buffer (pH 5.0, I=150 mM). Figure 5e shows some representative force profiles collected

10

in different buffers. The large variance between different force curves was probably because they

11

were recorded on different rBalcp19k fiber aggregates with different thickness and moduli owing

12

to different extent of aggregation. We did not collect force curves from the controls, because the

13

adsorbed protein layers of the controls are much thinner than that of rBalcp19k fiber aggregates

14

(a few nanometers versus several hundred nanometers), which makes the comparison ineffective.

15

Figure 5f shows the normalized adhesion force and adhesion energy of rBalcp19k nanofibers

16

from three independent experiments (Refer to Table S4 for detailed results). Due to different

17

experimental setup, we cannot directly compare the adhesion force of rBalcp19k nanofibers with

18

that of rBalcp19k monomers. Notably, we found that self-assembled rBalcp19k nanofibers and

19

monomeric rBalcp19k showed significantly different adhesive property: the adhesion ability of

20

rBalcp19k monomers decreased with increased pH (Figure 3d), whereas the adhesion ability of

21

rBalcp19k nanofibers increased when solution pH became higher (Figure 5f). Importantly, the

22

adhesion of rBalcp19k nanofibers could resist the adverse impact of high-salinity seawater (pH

23

8.0, I=550 mM). Moreover, we noticed that in seawater the rupture corresponded to cohesive


20

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1

rather than adhesive failure, indicating that the real adhesion force of rBalcp19k nanofibers was

2

larger than the values in Figure 5f. In summary, we confirmed that pre-assembled rBalcp19k

3

nanofibers were stable in seawater and showed improved adhesion ability. Previously, Zhong et

4

al. evaluated the adhesion ability of amyloid structures constructed by fusing CsgA (a major

5

subunit of E. coli curli fibers) and mussel adhesive proteins.37 While there exist many differences

6

in the test parameters, compared to the multi-protein nanofibrils, whose underwater adhesion

7

energy reached ~20 mJ m-2, rBalcp19k nanofibers exhibited much lower adhesion strength.

8

However, both studies indicated that when adhesive protein monomers self-assembled into

9

ordered nanostructures, their adhesion ability would be greatly improved and show resistance to

10

the adverse environmental conditions, e.g., mussel foot proteins in the multi-protein nanofibers

11

exhibited better oxidation tolerance,37 while the adhesion of rBalcp19k nanofibers could resist

12

the adverse effects of high-salinity seawater.

13


21


Page 22 of 32

1

Figure 5. Characterization of the morphology and adhesion ability of pre-assembled rBalcp19k

2

nanofibers in seawater. (a) An AFM phase image showing pre-assembled rBalcp19k nanofibers

3

in a pH 5.0, I=150 mM buffer at 4°C for one month. Self-assembled rBalcp19k nanofibers were

4

incubated in simulated seawater conditions (pH 8.0, I=550 mM) for one month at 4°C, and their

5

morphologies were examined after (b) 1 day, (c) 3 days and (d) 30 days. (e) Representative force

6

profiles collected on rBalcp19k fiber aggregates adsorbed on mica under different conditions. (f)

7

Normalized adhesion force and adhesion energy of rBalcp19k nanofibers at various conditions.

8

The figure showed the results from three independent experiments of 6×20 force curves.

9

The self-assembly of nanostructures to boost the underwater adhesion strength has not been

10

revealed in well-studied mussel and tubeworm adhesive proteins, suggesting that barnacles may

11

have evolved a unique strategy distinct from DOPA chemistry for strong underwater attachment.

12

We propose that by self-assembling into nanofibers, the cohesive interactions of cp19k are

13

greatly enhanced, which can compensate the electrostatic repulsion among likely charged lysine

14

residues in seawater and avoid cohesive failure. Furthermore, self-assembly may also lead to

15

dramatically improved interfacial hydrophobic interactions by the cooperation between

16

neighboring positive charges and hydrophobic domains, in a size and chemical environment

17

dependent manner.48, 49 It is worth noting that so far it is unclear which segment of rBalcp19k is

18

responsible for the self-assembly. It is likely that the low-complexity STGA-rich block in

19

rBalcp19k (Figure 1d) is the self-assembly motif, since it is homologous to the sequences of silk

20

proteins.9 The other block, which is abundant in hydrophobic and charged amino acids (Figure

21

1d), probably play a major role to bind to diverse substrates via van der Waals interactions.1, 10

22

Based on the structural characteristics of Balcp19k, we have synthesized a series of peptide

23

blocks. In the following studies, the self-assembly property and adhesion ability of these peptides


22

Page 23 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

will be explored. Moreover, in spite of recent advances in our knowledge about barnacle cement

2

glands,50-53 the localization and secreting mechanisms of barnacle cement proteins are not yet

3

fully understood. Consequently, the question that the detected amyloid-like fibrils at barnacle

4

adhesive interface are self-assembled before or after secretion remains unclear.54, 55 Combined

5

with future studies on barnacle physiology, the in vitro pH-dependent self-assembly behavior of

6

cp19k revealed in this study may provide new insights.

7

CONCLUSION

8

We found that the secondary structure of rBalcp19k was dominated by random coils and β

9

sheets, and it exhibited time-dependent transition behavior under low-pH conditions. For the first

10

time, we confirmed that rBalcp19k showed pH-dependent self-assembly behavior. Under acidic

11

and low-ionic strength conditions, rBalcp19k self-assembled into ThT-insensitive nanofibers,

12

probably via the silk proteins homologous blocks. Pre-assembled rBalcp19k nanofibers were

13

stable in seawater and did not disassemble. Without self-assembly, the adhesion ability of

14

rBalcp19k was weaker than that of unoxidized Cell-Tak, a mixture of DOPA-containing mussel

15

adhesive proteins, whereas after self-assembly, rBalcp19k nanofibers demonstrated improved

16

adhesion ability and significantly different adhesion property. Self-assembled rBalcp19k

17

nanofibers showed stronger while un-assembled rBalcp19k monomers showed weaker adhesion

18

when solution pH and ionic strength increased. Our results may provide new insights to

19

understand barnacle underwater attachment mechanism and design barnacle-inspired functional

20

materials.

21

ASSOCIATED CONTENT

22

Supporting Information

23

The following files are available free of charge.


23


1

Amino acid sequences, adhesion force and adhesion energy of rBalcp19k and control proteins,

2

force curves of the controls, MALDI-TOF mass spectroscopy, CD spectra and different

3

secondary structure contents of rBalcp19k, the self-assembly dynamics of rBalcp19k, and

4

additional AFM images of rBalcp19k and its mutant (PDF)

5

AUTHOR INFORMATION

6

Corresponding Authors

7

*E-mail: [email protected]

8

*E-mail: [email protected].

9

Author Contributions

Page 24 of 32

10

The manuscript was written through contributions of all authors. All authors have given approval

11

to the final version of the manuscript.

12

Notes

13

The authors declare no competing financial interest.

14

ACKNOWLEDGMENT

15

This work was supported by the National Natural Science Foundation of China (Nos. 21522402

16

and 11304156). C.L. thanks the China Scholarship Council (CSC) for offering a scholarship (No.

17

201503170277).

18

REFERENCES

19 20

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SYNOPSIS

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Self-Assembled Nanofibers for Strong Underwater Adhesion: The

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