Universidad Nacional de Chimborazo

NOVASINERGIA 2018, Vol. 1, No. 2, junio-noviembre (90-99)

ISSN: 2631-2654

https://doi.org/10.37135/unach.ns.001.02.10

Research Article

http://novasinergia.unach.edu.ec

Wet Adhesive Properties of Asian Green Mussel (Perna viridis) Foot

Protein Pvfp-5: An Underwater Adhesive Primer

Propiedades Adhesivas Húmedas de la Proteína del Pie de Mejillones Verdes

Asiático (Perna viridis) Pvfp-5: Una Base Adhesiva Subacuática

Navinkumar J Patil

*, Paola Gabriela Vinueza Naranjo

, Bruno Zappone

Dipartimento di Fisica, Università della Calabria, Arcavacata di Rende (CS), 87036, Italy.

DIET, Sapienza Università di Roma, Roma (RM), Italy.

Consiglio Nazionale delle Ricerche, Istituto di Nanotecnologia (CNR-Nanotec), Rende (CS) 87036, Italy;

paola.vinueza@uniroma1.it; bruno.zappone@cnr.it

* Correspondence: navinjpatil@gmail.com

Recibido 30 octubre 2018; Aceptado 28 noviembre 2018; Publicado 10 diciembre 2018

Abstract:

Asian green mussels (Perna viridis) are bivalves that attach firmly to rocks in wave-battered

intertidal seashores via a proteinaceous secretion. P. viridis mussels follow a precisely time-

regulated secretion of adhesive proteins where P. viridis foot protein-5 (Pvfp-5) was identified as

the first protein to be secreted during the formation of adhesive plaque. The high content of

catecholic amino acid 3,4-dihydroxy-L-phenylalanine (DOPA) (11 mol%) and cysteine (Cys)

(15 mol%) in Pvfp-5 and its localization near the plaque-substrate interface have prompted

speculation that the vanguard protein Pvfp-5 plays a key role as an adhesive primer in underwater

adhesion of P. viridis mussels. Surface Force Apparatus (SFA) was used to probe the adhesive

properties of Pvfp-5 at the nano-scale where pH dependent wet adhesion and antioxidant activity

of foot-extracted and purified Pvfp-5 were investigated. The study revealed that Pvfp-5 with its

high DOPA and CYS-content maintains adhesion even at higher pH by overcoming the

spontaneous oxidation of DOPA to quinone. SFA results are consistent with the apparent function

of Pvfp-5 acting as an adhesive primer, overcoming repulsive hydration forces by displacing

surface-bound water and generating strong and durable surface adhesion. Our findings reveal

molecular-scale insights that should prove relevant and impact material sciences to help the

development of new generation of wet-resistant adhesives, coatings and glues for biomedical,

therapeutic and antifouling applications.

Keywords:

Adhesion, Cysteine (CYS), Dihydroxy-L-phenylalanine (DOPA), Mussel foot proteins (Mfps),

Perna viridis foot protein (Pvfp), Surface Forces Apparatus (SFA)

Resumen:

Los mejillones verdes asiáticos (Perna viridis) son bivalvos que se adhieren firmemente a las rocas

en las costas intermareales golpeadas por las olas a través de una secreción proteica. Los

mejillones de P. viridis siguen una secreción de proteínas adhesivas regulada en el tiempo, donde

la proteína del pie-5 de P. viridis (Pvfp-5) fue identificada como la primera proteína que se secreta

durante la formación de la placa adhesiva. El alto contenido de aminoácido catecólico 3,4-

dihidroxi-L-fenilalanina (DOPA) (



11% en moles) y cisteína (Cys) (



15% en moles) en Pvfp-5 y

su localización cerca de la interfaz placa-sustrato tienen provocó la especulación de que la

proteína de vanguardia Pvfp-5 desempeña un papel clave como imprimación adhesiva en la

adhesión bajo el agua de los mejillones de P. viridis. El aparato de fuerza de superficie (SFA) se

usó para probar las propiedades adhesivas de Pvfp-5 a escala nanométrica, donde se investigó la

adhesión húmeda dependiente del pH y la actividad antioxidante de Pvfp-5 purificado y extraído

con el pie. El estudio reveló que Pvfp-5 con su alto contenido de DOPA y CYS mantiene la adhesión

incluso a un pH más alto al superar la oxidación espontánea de DOPA a quinona. Los resultados

de SFA son consistentes con la función aparente de que Pvfp-5 actúa como imprimación adhesiva,

superando las fuerzas de hidratación repulsivas al desplazar el agua unida a la superficie y

generando una adhesión superficial fuerte y duradera. Nuestros hallazgos revelan conocimientos

a escala molecular que deberían ser relevantes e impactar en las ciencias de los materiales para

ayudar al desarrollo de la nueva generación de adhesivos, recubrimientos y pegamentos resistentes

a la humedad para aplicaciones biomédicas, terapéuticas y antiincrustantes.

Palabras

clave:

Adhesión, Cisteína (CYS), Dihidroxi-L-fenilalanina (DOPA), Proteínas del pie de mejillón (Mfps),

Perna viridis proteína del pie (Pvfp), Aparato de fuerzas de superficie (SFA).

http://novasinergia.unach.edu.ec 91

1 Introduction

Animal attachment to a substrate is very different

in terrestrial and aquatic environments. In

terrestrial environment, gravity is considered as

the most important detachment force. However, in

submerged conditions gravity is nearly balanced

out by buoyancy but flow forces such as drag and

lift are of higher importance (Ditsche et al. 2014).

As is well known, water or moisture significantly

compromises the performance of most man-made

adhesives, as water effectively competes for

surface bonding and eliminates contributions of

van der Waals interactions (Comyn, 1981; Lee et

al. 2011).

Despite these prevalent challenges and the

harshness of the physical environment, wave-

swept rocky shores and ship hulls are home to a

variety of organisms that have evolved to attach

themselves permanently to variety of substrates

that are wet, saline, corroded, and/or fouled by

biofilms. Figure 1 shows variety of invertebrate

animals attached underwater to wave-swept rocks

and ship hulls that have evolved separate and

distinct working strategies for their particular

underwater bonding requirements to protect

themselves against biological, chemical, and

mechanical stresses from the ocean (Wilker, 2010;

Stewart et al. 2011).

Figure 1: Mussels, barnacles, and tube worms sticking

to rocks (A-C); a starfish is shown adhering to a sheet of

glass (D). Figure adopted from (Wilker, 2010).

Mussels are prominent examples of sessile type

organisms attaching themselves permanently to

variety of underwater solid substrates. A detailed

study on Mytilus genus (blue mussels) during the

last decade has enhanced our understanding of

underlying biochemical and biophysical

adaptations of mussels for opportunistic and

durable adhesion to moist mineral and metal oxide

surfaces (Yu et al. 2011a; Wei et al. 2013; Yu et

al. 2013). Mussel adhesion is mediated by a

holdfast structure known as the byssus, essentially

a bundle of macroscopic extensible high-

performance fibers tipped by flattened adhesive

plaques that tether the mussel firmly to a variety of

hard surfaces and play a critical role in the ability

of mussels to dominate space on many temperate

shores worldwide (Waite et al. 2005; Lin et al.

2007; Priemel et al. 2017). Byssal thread consists

of several collagen-like proteins responsible for the

mechanical properties of the fibers while the plaque is

composed of number of protein-based adhesives

containing 3,4-dihydroxyphenylalanine (DOPA),

a post translationally modified amino acid that

forms hydrogen bonds with almost any surface

chemistry under wet conditions. Mussels adhere to

surfaces underwater through the secretion,

deposition, and complexation of these DOPA-

containing proteins, known as mussel foot proteins

(Mfps), to resist the forces produced by crashing

waves – a feat unmatched by current industrial

glues (Priemel et al. 2017; Hamada et al. 2017).

Figure 2: Cartoon of mussel byssus and proposed model

of mussel’s adhesion mechanism. (A) schematic

overview of a mussel; (B) schematic of Mfps delivery

from different precursors under mussel’s foot; (C) Mfp

incremental secretion: step 1, acid; step 2, primers (mfp-

3 and mfp-5); step 3, reducing agent (mfp-6); step 4,

bulk adhesives (mfp-2 and mfp-4) and collagen; and step

5, coating proteins (mfp-1); (D) schematic showing the

distribution of major mfp’s and collagen in byssal

plaque. Figure adopted from (Kollbe Ahn, 2017).

Within the adhesive plaque of blue mussels

(Mytilus species), six major mussel foot proteins

(Mfp’s 1-6) have been identified with substantially

different amino acid sequences which are

responsible for adhesion and coating of the mussel

byssus (Kollbe Ahn et al. 2017; Lu et al. 2013).

Each Mfp is found at a different location in the foot

and byssus, and each one is believed to play a

distinct and important role, as shown in figure 2

(Ahn et al. 2017; Silverman at al., 2007; Hwang et

al. 2010). Mfps are polyelectrolytes distinguished

by their isoelectric point (pI ∼ 10) and mussels

appear to gain surface access for their adhesive

proteins by using their own protein-based ions to

outcompete the ions in their saltwater environment

http://novasinergia.unach.edu.ec 92

(Wilker, 2015). Also, the adhesive proteins found

near the plaque-substrate interface are the proteins

with the highest amount of DOPA, thus prompting

the important and involvement of DOPA in surface

adhesion. DOPA contains a chemical group called

catechol (3,4-dihydroxyphenyl), made from a

benzene ring bearing two adjacent hydroxyl (-OH)

groups, as shown in figure 3. Further studies on

catecholic amino acid revealed that the hydroxyl

groups of the catechol form chemical bonds with

rocks and other substrates that help to stick the

mussel in place (Ornes, 2013).

Figure 3: Dopamine and DOPA showing catechol

moiety.

Although DOPA-mediated wet adhesion is strong

and versatile on different surfaces, it has a

troubling tendency to readily oxidize to Dopa-

quinone and formation of Dopaquinone diminishes

adhesion of Mfps to surfaces by at least 80-95%.

There are several factors which may lead to

oxidation of DOPA to Dopa-quinone.

Spontaneous oxidation occurs at alkaline pH and

auto-oxidation which refers to spontaneous

oxidation of cathechol in the presence of oxygen at

neutral pH leads to loss of adhesion of Mfps (Lee

et al. 2006; Menyo et al. 2013). Regulation of

redox in the adhesive byssus of marine mussels is

an exotic and fascinating case in point of

extracellular redox where higher cysteine (CYS)

content in some Mfps reduces the risk of DOPA

oxidation.

Recently identified byssal plaque proteins from the

Asian green mussel Perna viridis known as Pvfps

are all enriched with cysteine (CYS) and DOPA-

residues unlike Mfps, where only particular variant

of plaque protein (e.g. Mfp-6) has high CYS

content which acts as an anti-oxidant, reducing

dopamine back to DOPA. Also, the primary

sequences of Pvfps share very low homology with

earlier studied Mfps (Petrone et al. 2015). Our

current knowledge on the nature and properties of

mussel adhesion is based only on the limited set of

mussel species studied. Therefore, understanding

the molecular basis of adhesion in green mussels

can lead us to alternate mussel inspired

bioadhesives and targeted anti-fouling strategies.

Natural foot proteins from mussels of P. viridis

were extracted by artificially inducing the live

mussels to secrete plaque-forming proteins which

were collected from the groove at the tip of the foot

organ (Petrone et al. 2015; DeMartini et al. 2017).

It was identified that the byssal plaque proteins

were secreted in time-regulated manner where

Perna viridis foot protein-5 (Pvfp-5) was the first

protein secreted by Asian green mussels to initiate

interaction with the substrate, displacing

interfacial water molecules and forming adhesive

bonds with the substrate which was then followed

by other variants of Pvfps. Pvfp-5, which typically

appeared after 10 s of saline injection, had

molecular weight (MW) in the range 8-10 kDa and

pI 9.2. Amino acid sequence of Pvfp-5 revealed

that it contained 12 CYS residues, accounting for

15 mol% of its amino-acid content. It also revealed

21 mol% Tyr side chains in its primary sequence

and 9 DOPA residues accounting for 11 mol%

DOPA in Pvfp-5. Pvfp-5 predominantly exhibited

random coil structure and the dynamic light

scattering (DLS) revealed a hydrodynamic

diameter, d

= 9.500.26 nm (Petrone et al. 2015).

Here we report on our investigation of the

attractive forces and work of adhesion between

thin films of purified Pvfp-5 under changing pH

conditions. The results support the conclusion that

distinct mussel species adopt different strategies

for underwater adhesion, where Pvfp-5 unlike

Mfp-5 maintains adhesion even at higher pH due

to its high DOPA and CYS contain and CYS-based

redox activity.

2 Methodology

2.1 Extraction and Purification of

Mussel Foot Protein

P. Viridis foot protein (Pvfp-5) was extracted from

the foot organ of P. viridis mussels collected from

the northern coast of Singapore. Methodology for

extraction and purification of Pvfp-5 has been

previously reported (Petrone et al. 2015). The

secretion of adhesive foot proteins of P. Viridis

mussels was triggered by injecting 1 ml of KCl

phosphate buffer (0.56M, pH 7.2) into the mussels

pedal nerve located at the base of the foot.

Injecting KCl solution in the foot organ reasonably

mimics the natural byssus secretions of P. viridis,

and is referred as saline-induced mussel secretion.

These secretions of P. viridis were then collected

from the groove at the tip of the foot organ.

2.2 Surface Forces Apparatus

(SFA)

Adhesion and normal force measurements

between Pvfp-5 layers adsorbed on smooth and

chemically inert surfaces of mica (a common

alumino-silicate clay mineral) were obtained using

http://novasinergia.unach.edu.ec 93

the a SFA Mark III (Surforce LLC, Santa Barbara,

CA, USA). Figure 4 shows a detailed schematic

diagram of the SFA set-up used in this study. The

SFA is a technique to measure the interaction

forces between two macroscopic surfaces as a

function of absolute distance between them using

multiple beam interferometry (MBI) and fringes of

equal chromatic order (FECO). A detailed

description of the technique can be found in

Israelachvili & McGuiggan (Israelachvili et al.

1990).

Figure 4: Schematic of the SFA set-up: crossed cylinder

experimental set-up of the SFA-MK III (A); resulting

fringe pattern of fringes of equal chromatic order

(FECO) (B). Pvfp-5 was adsorbed on both mica surfaces

at pH 3 for 20 minutes followed by rinsing with protein-

free buffers of variable pH to study the effect of pH on

cohesive interaction between Pvfp-5 molecules.

All the SFA experiments were carried out using

mica as the base surface. Since the freshly cleaved

thin sheets of mica are atomically smooth,

optically transparent, and chemically inert, they act

as ideal substrates for SFA experiments to study

adhesive properties of Pvfp-5 proteins. The outer

opposing sides of mica sheets were coated with a

semitransparent layer of silver ( 55 nm) using

thermal evaporation or chemical sputtering

technique. Identically thick and flat back-slivered

mica sheets (thickness  1-5 µm) were glued with

the silver coated side on the cylindrical glass disks

(radius of curvature, R=1.8–2.2 cm) using

thermosetting glue (EPON 1007 by Shell). Two

semi-transparent curved disks were then mounted

orthogonally to each other in an inert nitrogen

atmosphere inside an airtight SFA chamber. The

top disk was fixed and the bottom disk was

attached to the free end of the double cantilever

spring where the stiffness ‘k’ of the spring was in

the range of 250-950 N/m. The spring and

displacement mechanism allowed the motion of

opposing disks into a well-defined single asperity

contact at a given force.

During the experiment, collimated white light is

guided through these disks which act as semi-

transparent mirrors. When the surfaces are moved

close together, together with the intervening

medium it forms a symmetrical three-layer Fabry-

Perot interferometer employing MBI. The

constructive and destructive interference of the

white light at discrete wavelengths leads to the

generation of the FECO, which are an infinite

series of alternating sharp bright and dark bands

which are then detected by the spectrophotometer.

A typical FECO is shown in figure 4B, which

clearly depicts the flat section of FECO, indicating

an extended flat zone of contact. The FECO allows

the determination of the inter-mirror distance with

a resolution below 1Å. During the initial contact

mica-mica contact in dry nitrogen before

adsorbing proteins, the distance D=0 was defined

by recording the set of wavelengths. A change in

FECO fringe position was used to calculate the

shift in distance (D), of two opposing mirrors,

where discrete spectral wavelengths of mercury

were used to calibrate the spectrograph.

The SFA experiments were performed by precisely

approaching the surfaces until they come in

adhesive contact followed by retracting them from

one another. The adhesive forces were then

calculated by using Hooke’s law (F=k×ΔD) where

k is the known stiffness of the spring on which

lower surface was mounted and ΔD is the shift in

distance from the contact (of adhesive surfaces) to

their point of separation. Because of the curved

geometry of the surfaces, the measured force was

normalized by the radius of curvature of the

surfaces (R). By using the Derjaguin

approximation, the normalized force F(D)/R can

be directly transformed into the interaction energy

per unit area between the two flat surfaces, which

is given by E(D) = F(D)/2



The error on F/R was smaller than 0.1mN/m (force

detection threshold) and the error on D about 0.5

nm.

2.3 Sample Preparation for SFA

Force Measurements

Mussel adhesive proteins like Pvfp-5 are

polyelectrolytes possessing ionization tendencies

which define their interactions. Therefore, careful

selection of pH was made during Pvfp-5

adsorption and for buffer solutions used for rinsing

the adsorbed proteins (Yu et al. 2011a; Danner et

al. 2012). Also, well defined salt concentration

was used to provide sufficient counterions to

prevent the electrostatic self-interaction from

dominating in the SFA experiments (Lin et al.

2007; Yu et al. 2011a; Danner et al. 2012; Lu et al.

2013).

Following mica-mica contact in dry air, Pvfp-5

proteins were adsorbed on both mica surfaces from

http://novasinergia.unach.edu.ec 94

0.02 mg/ml solution of protein in acid saline buffer

containing 10 mM acetic acid (AcOH) and 0.25M

potassium nitrate (KNO

) in purified water (Milli-

Q grade from Millipore) at pH 3. After a 20 min

adsorption, the surfaces were rinsed with protein-

free acidic buffer solution. Following positioning

into the SFA, the surfaces were brought almost

into contact with a droplet of buffer solution

providing a bridge between the two surfaces. To

keep the buffer droplet from evaporating, a second

droplet of buffer was placed in the box to maintain

the vapor pressure.

Figure 5: Representative force-distance (F–D) profile: (A) Normalized force F/R measured between two Pvfp-5 coated

mica surfaces at pH 3. Positive forces are repulsive and negative forces are attractive. (B) Semi-logarithmic plot

showing the range of repulsion 2T, force detection threshold (dotted line) and exponential curves F/R=Ae

-D/d

. Solid

symbols (●, ●, ■ and ■) indicate forces on approach and open symbols (○, ○, □ and □) indicate forces on

retraction of surfaces. Each symbol (circle and square) corresponds to different contact position and different colors

indicate different surface approach/retraction cycles.

The influence of pH was investigated by flushing

the droplet of acidic buffer (~100 µL) with an

excess of the new treatment buffer (3ml) with

neutral and basic pH. Treatment buffers were

1) Neutral saline buffer, 0.25M KNO

, pH ≈ 7

(without HCl or NaOH)

2) Alkaline/basic saline buffer, 0.25M KNO

, pH

≈ 10 (with NaOH)

KNO

was used to replace sodium chloride in the

buffer solution to avoid corrosion of the thin silver

layers under the mica substrates. Having high

concentrations of chloride ions in solution, affect

the quality of the optical fringes during the SFA

experiments. Degassed buffer solutions were used

for dissolving Pvfp-5 as well as for rinsing.

Degassing helps removal of dissolved gases in

solution and minimizes the occurrence of bubbles

between two surfaces while performing SFA

experiments. Multiple force runs were recorded for

each pair of mica and protein adsorption and SFA

force measurements were done at a fixed

temperature of 25±1 C.

3 Results and Discussion

Pvfp-5 is the first protein to be secreted by Asian

green mussels (Perna viridis) in the precisely time

regulated secretion of a series of adhesive proteins.

Thus, Pvfp-5 is the first protein to initiate contact

with the substrate which acts as an adhesive

primer. Pvfp-5 is enriched with cysteine and

tyrosine (Tyr)/ DOPA residues and its localization

at plaque-substrate interface have provoked

interest in its adhesive properties. Using SFA, the

effects of changing pH on adhesion capability of

Pvfp-5 was explored. Since Pvfp-5 was adsorbed

onto both the opposing mica surfaces, the adhesion

forces reported below are proportional to the

cohesive interaction between Pvfp-5 molecules.

3.1 Adhesion between Pvfp-5

Layers at Acidic pH

Interaction forces (F/R) between the thin layers of

Pvfp-5 adsorbed symmetrically to both the mica

surfaces at acidic pH were measured as a function

of separation distance (D) (figure 5A). Seven

distinct force runs were performed at three

different contact positions where the surfaces were

brought into contact in the acid saline buffer and

then separated after a brief contact time. At pH 3

and 0.25M ionic strength, the maximum adhesion

force after separation was measured to be F

/R =

5.42 mN/m, which can be converted to adhesion

energy, E = (2/3



) F

/R ≈ 1.14 mJ/m

. Different

force runs displayed variability in adhesion force

since factors like contact time or maximum load

applied during compression were not explicitly

controlled. However, a clear trend emerged that

adhesion decreased with time as the surfaces were

repeatedly approached and retracted at the same

contact position and as different contact positions

were tested. This was most likely due to oxidation

of DOPA (Ahn et al. 2014; Nicklisch et al. 2012).

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The average value of the adhesion force was F/R =

3.63 mN/m. The repulsive force was recorded for

all the force runs during surface approach, showing

that adhesion was generated by the formation of

adhesive bonds between interacting Pvfp-5

molecules. The force consistently exceeded the

detection threshold of 0.1 mN/m when the surface

separation distance D became smaller than 2T =

24.5–30.8 nm (figure 5B), where T was the

thickness of adsorbed protein layer on one surface.

Since the Debye length of the saline buffer was

smaller than 1 nm, the recorded long repulsion

range (2T) can be attributed to the overlap between

protein layers adsorbed on opposite surfaces, each

having an approximate thickness T = 12.2–15.4

nm. The value of the thickness of adsorbed protein

layer on mica was larger than the size of Pvfp-5

molecule determined by DLS (Petrone et al. 2015),

indicating aggregation behavior of Pvfp-5

molecules at pH 3. The aggregation of Pvfp-5

molecules in solution was also confirmed by

dynamic light scattering (DLS) results indicating

the hydrodynamic diameter of Pvfp-5 to be 8.56

nm at pH 4, which was most likely via DOPA–

DOPA crosslinks.

The maximum value thickness (T) of a Pvfp-5

layer on mica substrate was obtained during the

first approach, indicating that Pvfp-5 was adsorbed

as a soft hydrated layer, as opposed to a compact

“hard-wall” coating, allowing the protein

molecules and water to rearrange and move as the

compressive force was increased. This also

indicates that the protein layers were flattened,

perhaps due to the formation of additional DOPA-

DOPA crosslinks or DOPA-mica hydrogen bonds

on compression. Besides, the determination of

thickness (T) of the adsorbed protein layer on mica

substrates, a “hard wall” distance (D

) was also

measured using SFA.

Hard wall distance is defined as the asymptotic

thickness of confined Pvfp-5 under increasing

normal load or pressure. Hard wall distance was

determined by compressing the adsorbed Pvfp-5

layer until FECO fringes cease to move towards

the lower wavelength side of the signal. The

measured hard wall distance for Pvfp-5 at pH 3

was reported to be D

≈ 2.6 nm.

Figure 6: Representative force-distance (F–D) profile: (A) Normalized force F/R measured between two Pvfp-5 coated

mica surfaces at neutral pH (=7). (B) Semi-logarithmic plot showing the range of repulsion 2T, force detection threshold

(dotted line) and exponential curves F/R=Ae

-D/d

. Solid symbols (●, ●, ■ and ■) indicate forces on approach and open

symbols (○, ○, □ and □) indicate forces on retraction of surfaces. Each symbol (circle and square) corresponds to

different contact position and different colors indicate different surface approach/retraction cycles.

3.2 Adhesion between Pvfp-5

Layers at Neutral pH

Interaction forces (F/R) between the thin layers of

Pvfp-5 at neutral pH were measured by first

adsorbing Pvfp-5 to both mica surfaces at acidic

pH, followed by rinsing the surfaces with a

protein-free neutral saline solution. At pH 7 and

0.25M ionic strength, the surfaces were brought

together and the maximum adhesion force

measured after separation was F

/R = 1.28 mN/m

(figure 6A), corresponding to the work of

adhesion, E = (2/3



) F

/R ≈ 0.27 mJ/m

. Maximum

adhesion was measured during separation of the

first approach-retraction cycle which consequently

reduced for the next three force runs recorded on

the same contact position.

This trend at pH 7 was similar to that observed at

pH 3 earlier, which was most likely due to DOPA

oxidation. The average value of the adhesion force

at pH 7 was F/R = 0.84 mN/m. Hysteresis was

present for all the force runs recorded and

repulsive forces were observed during surface

approach with the onset of repulsion at 2T = 36–

45.8 nm (figure 6B). Thin hard-wall distance, D

≈ 2.2 nm was measured at neutral pH.

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3.3 Adhesion between Pvfp-5

Layers at Basic pH

To assess the interaction forces (F/R) between

Pvfp-5 layers at basic pH, thin layers of Pvfp-5

were initially adsorbed on both mica surfaces at

acidic pH, followed by rinsing with protein-free

alkaline buffer solution. Two force runs were

recorded at pH 10 and 0.25M ionic strength and

small adhesion were measured for both the force

runs. The maximum adhesion force measured at

pH 10 was 0.68 mN/m (figure 7A), corresponding

to a work of adhesion, E = 0.14 mJ/m

The average value of the adhesion force at pH 10

was F/R = 0.6 mN/m. The approaching branch of

force curves revealed a repulsion setting in at a

comparably smaller surface separation of 2T =

11.4–29.4 nm than that recorded for acidic and

neutral pH (figure 7B). The measured hard wall

distance for Pvfp-5 at pH 10 was reported to be

≈ 3.85 nm.

3.4 Discussion

Mytilus edulis foot protein-5 (Mefp-5) is one of the

adhesive proteins in the byssal adhesive plaque of

the mussels of M. edulis that has been reported to

have highest molecular DOPA concentration

which is localized near the plaque substrate

interface similar to Pvfp-5 (Danner et al. 2012,

Petrone et al. 2015). The adhesion capabilities of

Pvfp-5 were assessed by SFA experiments using

mica as the substrate, which enabled direct

comparison of Pvfp-5 with extensively studied

Mefp-5. Strong adhesion force was consistently

measured between symmetrically adsorbed Pvfp-5

films at pH 3 (maximum adhesion force, F

/R =

5.42 mN/m). Previous studies on Mefp-5 showed

adhesion force, F

/R = 6.0 mN/m, which slightly

exceeded the adhesion force of Pvfp-5.

Considering the 30 mol% DOPA content of Mefp-

5 compared to 11 mol% DOPA in Pvfp-5, the

adhesion force between interacting proteins films

of both the adhesive proteins were not significantly

different (Danner et al. 2012).

It has been reported that exposure of Mefp-5 to pH

above 7.5 reduces its adhesion by 95% due to rapid

oxidation of DOPA to Dopaquinone (Kan et al.

2014). However, the CYS-enriched Pvfp-5 was

adhesive at both neutral and basic pH with the

maximum adhesion force, F

/R = 1.28 mN/m at pH

7.0 and F

/R = 0.68 mN/m at pH 10 (table 1).

Thus, increasing the pH of gap solution from pH3

to pH7 and pH10, reduced the adhesion force by

approximately 75% and 87%, respectively,

compared to initial adhesion force measured at

acidic pH. Therefore, CYS residues in the amino

acid sequence of Pvfp-5 participate in the

reduction of quinones by providing a pH-

dependent antioxidant activity to Pvfp-5, the first

protein secreted by P. viridis, justifying its role as

a vanguard protein that acts as an adhesive primer

which initiates first adhesive interactions with the

substrate.

Besides adhesion, table 1 summarizes the variation

of other parameters with respect to change in pH

conditions which reveals an important trend: the

hard wall and the thickness (T) of Pvfp-5 layer on

mica, changed with pH.

Table 1: Summary of various parameters for Pvfp-5

interactions in symmetric configuration measured by

SFA.

Max.

adhesion

force,

(mN/m)

Max.

adhesion

energy,

(mJ/m

)

Pvfp-5 film

thickness,

T (nm)

Hardwall

distance,

(nm)

pH 3

5.42

1.14

12.2 – 15.4

2.6

pH 7

1.28

0.27

18 – 22.9

2.2

pH 10

0.68

0.14

5.7 – 14.7

3.8

The film thickness (T) of Pvfp-5 on mica at pH 3

was in the range 12.2-15.4 nm with hard wall, D

(pH 3) ≈ 2.6 nm. After increasing the pH of the gap

solution to 7.0, repulsion at comparatively higher

distance was noticed during approach. The film

thickness (T) at pH 7 was measured to in the range

of 18-22.9 nm with hard wall, D

(pH 7) ≈ 2.2

nm. The increase in film thickness at pH 7 suggests

that the Pvfp-5 film expands at neutral pH;

however, the hard wall distance measured on

compression remained almost unchanged. When

the pH of the gap solution was changed from acidic

to alkaline, significantly low repulsion range

corresponding to film thickness (T) in the range

5.7-14.7 nm was recorded. Surprisingly, the hard

wall distance measured upon compressing the

Pvfp-5 layer under higher loads was D

(pH 10)

= 3.8 nm, which was higher than D

recorded at

pH 3 and pH 7, respectively. Lower film thickness

and higher hard wall at pH 10 suggests a more

compact and rigid conformation of Pvfp-5, which

may prevent the exposure of DOPA residues to

higher pH, thus preventing DOPA from

spontaneous oxidation.

Surprisingly, SFA experiments on Pvfp-5 also

showed that adsorption of thicker layers of protein

on mica resulted in diminished adhesion (results

not shown). Series of experiments performed with

varying adsorption time and protein concentration

in solution revealed that thinner layer have more

exposed DOPA molecules available to interact and

form adhesive bonds whereas, thicker layers

screen DOPA molecules as well as enhances cross-

linking between DOPA molecules making them

unavailable for subsequent adhesive interactions.

http://novasinergia.unach.edu.ec 97

Figure 7: Representative force-distance (F–D) profile: (A) Normalized force F/R measured between two Pvfp-5 coated

mica surfaces at pH 10. (B) Semi-logarithmic plot showing the range of repulsion 2T, force detection threshold (dotted

line) and exponential curves F/R=Ae

-D/d

. Solid symbols (● and ●) indicate forces on approach and open symbols (○

and ○) indicate forces on retraction of surfaces. Different colors indicate different surface approach/retraction cycles.

The film thickness (T) of Pvfp-5 on mica at pH 3

was in the range 12.2-15.4 nm with hard wall, D

(pH 3) ≈ 2.6 nm. After increasing the pH of the gap

solution to 7.0, repulsion at comparatively higher

distance was noticed during approach. The film

thickness (T) at pH 7 was measured to in the range

of 18-22.9 nm with hard wall, D

(pH 7) ≈ 2.2

nm. The increase in film thickness at pH 7 suggests

that the Pvfp-5 film expands at neutral pH;

however, the hard wall distance measured on

compression remained almost unchanged. When

the pH of the gap solution was changed from acidic

to alkaline, significantly low repulsion range

corresponding to film thickness (T) in the range

5.7-14.7 nm was recorded. Surprisingly, the hard

wall distance measured upon compressing the

Pvfp-5 layer under higher loads was D

(pH 10)

= 3.8 nm, which was higher than D

recorded at

pH 3 and pH 7, respectively. Lower film thickness

and higher hard wall at pH 10 suggests a more

compact and rigid conformation of Pvfp-5, which

may prevent the exposure of DOPA residues to

higher pH, thus preventing DOPA from

spontaneous oxidation.

Surprisingly, SFA experiments on Pvfp-5 also

showed that adsorption of thicker layers of protein

on mica resulted in diminished adhesion (results

not shown). Series of experiments performed with

varying adsorption time and protein concentration

in solution revealed that thinner layer have more

exposed DOPA molecules available to interact and

form adhesive bonds whereas, thicker layers

screen DOPA molecules as well as enhances cross-

linking between DOPA molecules making them

unavailable for subsequent adhesive interactions.

4 Conclusion

In summary, the objective of this work was

understanding the molecular interactions between

Pvfp-5 proteins adsorbed on mica substrates at

different pH conditions using the SFA technique.

The results provide important insights into the wet

adhesion mechanism of Asian green mussels (P.

Viridis), whereby a DOPA-rich primer (Pvfp-5) is

initially secreted because of its superior adsorptive

and adhesive abilities to interact with foreign

surfaces. Other experimental studies of Pvfp-5

shows the ability of Pvfp-5 to act as an adhesive

primer to displace surface-bound water from

hydrophilic surfaces allows the formation of strong

and durable bonds via its adhesive DOPA residues

exposed on the protein surface, hence creating an

environment conducive to the assembly of other

plaque components (Petrone et al. 2015).

By studying the effects of changing pH on

adhesive properties of Pvfp-5, the emerging

picture is that Pvfp-5 with its high DOPA and

CYS-content, maintains adhesion even at higher

pH and acts as a self-antioxidant, overcoming the

spontaneous oxidation of DOPA to quinone at

higher seawater pH ( 8) with saturating levels of

dissolved oxygen. Also, tyrosine, DOPA and

cysteine motifs in Pvfp-5 play a key role in

enabling the robust underwater adhesion exhibited

by P. viridis. More broadly, the study provided a

molecular basis for understanding the impressive

underwater adhesion of marine organisms, an

insight that should prove relevant for diverse areas,

from designing of mussel inspired bioadhesives to

engineering targeted anti-fouling strategies.

DOPA- and CYS- containing proteins like Pvfp-5

are crucial for wet adhesion in mussels that retards

oxidation by shielding the amino acids from the

solvent and endowing the protein with the ability

to maintain adhesion at neutral as well as basic pH

levels (Kaushik et al. 2015). Recent studies on the

underwater adhesion mechanisms of marine

fouling organisms have been explored by SFA,

where three key mechanisms of successful

underwater adhesion, i.e. DOPA and REDOX

chemistry, cation-π interactions and complex

coacervation, have been suggested (Hwang et al.

2010; Yu et al. 2011; Gebbie et al. 2017). SFA and

other techniques have not yet fully elucidated all

the adhesion mechanisms of the marine fouling

organisms like mussels, but the key mechanisms

http://novasinergia.unach.edu.ec 98

exploited should be effective strategies for the

development of bio-inspired medical adhesives

and tissue sealants, antifouling surfaces for

medical devices and environmentally friendly

antifouling paints for use in the marine industry.

Further, understanding the aspects of natural redox

control can provide fundamentally important

insights for adhesive polymer engineering, surface

modifications and antifouling strategies.

Interest Conflict

The authors declare that they have no conflict of

interest.

Acknowledgment

We would like to thank Prof. Ali Miserez (NTU,

Singapore) for providing the isolated and purified

mussel foot proteins. This research was supported

by the University of Calabria (Unical), Italy

through its doctoral fellowship to N. J. Patil under

Bernardino Telesio School of Science and

Technique. We thank CNR-Nanotec and

Department of Physics, Unical for various

experimental and computing facilities.

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