INTEGR. COMP. BIOL., 42:1172–1180 (2002)
Adhesion à la Moule1
J. HERBERT WAITE2
Marine Science Institute, Department Molecular, Cell & Developmental Biology, University of California,
Santa Barbara, California 93106
SYNOPSIS. Mussels owe their sessile way of life in the turbulent intertidal zone to adaptive adjustments in
the process and biochemistry of permanent attachment. These have understandably attracted scientific interest given that the attachment is rapid, versatile, tough and not subverted by the presence of water. The
adhesive pads of mussel byssus contain at least six different proteins all of which possess the peculiar amino
acid 3, 4-dihydroxyphenylalanine (DOPA) at concentrations ranging from 0.1 to 30 mol %. Studies of protein
distribution in the plaque indicate that proteins with the highest levels of DOPA, such as mefp-3 (20 mol
%) and mefp-5 (30 mol %), appear to predominate at or near the interface between the plaque and substratum. Although the presence of DOPA in proteins has traditionally been associated with cross-linking via
chelate-mediated or covalent coupling, recent experiments with natural and synthetic DOPA-containing
polypeptides suggest that cross-link formation is not the only fate for DOPA. Intact DOPA, particularly
near the interface, may be essential for good chemisorption to polar surfaces. Uniformly high DOPA oxidation to cross-links leads to interfacial failure but high cohesive strength, while low DOPA oxidation results
in better adhesion at the expense of cohesion. Defining the adaptations involved in balancing these two
extremes is crucial to understanding marine adhesion.
There is an age of innocence in the pursuit of every
scientific discipline. After almost 2,500 years, the science of bioadhesion in most respects has yet to emerge
from it. With very few exceptions, most examples of
bioadhesion have yet to be reduced to a simple set of
principles in chemical and mechanical engineering.
Mussel attachment was the subject of one of the earliest recorded observations of bioadhesion. Aristotle
(transl. 1910) noted that the holdfast in the fan mussel
(Pinna) consisted of a robust bundle of fibers with
sticky tips. The term byssus (Greek bysso§ for flax
linen) was accidentally coined by him for the holdfast
(van der Feen, 1949) and has since gained universal
acceptance. As a model system for bioadhesion, byssal
attachment has always had popular appeal. Mussels are
common and their attachment is easy to watch; formation of byssus is rapid, robust, and relatively simple
in that proteins are secreted into the ventral groove of
the foot and molded there prior to release as a new
byssal thread. Further details on byssus formation
mostly in the blue mussel, Mytilus edulis, can be found
in numerous reviews on the subject (Brown, 1952;
Price, 1983; Waite, 1983, 1992).
Byssal appeal also has a technological cachet in that
mussels attach their byssus to virtually any solid surface. Such versatility intrigues engineers. Add to this
the fact that adhesive bonding is occurring in a turbulent saline environment, and it assumes much more
than a passing interest. It is appropriate perhaps to
point out that water poses some serious challenges to
adhesive bonding, and many manufacturing processes
go to great lengths to exclude water from surfaces to
be adhesively bonded (Cayless, 1991). The chemistry
of industrial adhesive bonding is generally of two
types: highly energetic (covalent or chelate) or a collection of weaker noncovalent interactions. While the
former can be exploited in specific cases where much
is known about the chemistry of the adhesive and adherend, most types of adhesion rely on extensive noncovalent interactions across the interface of the adhesive and adherend. These interactions include chargecharge, hydrogen bond, dipole-dipole, induced dipoledipole, and nonpolar couplings, among others. The
latter three are among those often grouped together as
van der Waals forces. Expressions for the interactive
energies of these interactions have been formulated for
all but the H-bonds and reflect that all depend inversely on the dielectric constant (or sometimes the square
of the dielectric) of the medium in which they occur
(Israelachvili, 1985). Given that the dielectric constant
of air is about 1 at 258C, in water at the same temperature it is about 80. In other words, other factors
being equal, the best interfacial interactions that an
adhesive might muster in water with an adherend,
would be only 1/80 what they are in air. Water, however, presents an additional challenge. The previous
scenario presupposes that adhesive molecules can actually make contact with the adherend surface. In those
instances where even this is not possible, water becomes a ‘‘weak boundary layer’’ (Schonhorn, 1981).
It has no monopoly as a spoiler of adhesion: dirt, oil,
grease, slime and oxides pose similar hazards. All
amount to a predicament similar to adhesive bonding
underwater. When there is no direct contact between
the adhesive and adherend, bond performance is at the
mercy of the cohesive strength within the boundary
layer itself. Thus even a residual boundary layer can
easily become the weakest ‘‘link in the chain’’ (Holubka et al., 1984). Given that they attach to hard surfaces underwater, mussels appear to be able to remove
1 From the Symposium Biomechanics of Adhesion presented at the
Annual Meeting of the Society for Integrative and Comparative Biology, 2–6 January 2002, at Anaheim, California.
2 E-mail: [email protected]
1172
MUSSEL ADHESION
1173
weak boundary layers including water from surfaces
and, once attached, their adhesion is not subverted by
the dielectric constant of the medium all around them.
This is remarkable for reasons cited if their adhesion
relies mostly on noncovalent interactions with the surface. If their adhesion also relies on more energetic
interfacial interactions, it is even more remarkable given the range of materials they attach to. Indeed, the
latter would suggest that they are able to recognize and
respond to different kinds of surfaces.
TABLE 1. Adhesive tensile strength of mussel byssal plaques
(adapted from Young and Crisp, 1982).*
ADHESIVE STRENGTH OF MUSSEL BYSSUS
There are reports of mussel tenacity, of byssal tensile strength, and of adhesive tensile strength. Some
caution is required to keep these concepts separate.
Tenacity denotes the force required to dislodge an attached mussel (Bell and Gosline, 1997). It is an interesting biological capability that necessarily focuses on
the whole animal but does not generally make adjustments for the varying numbers of threads present in
each animal. The tensile strength of a byssal thread
pertains to the length of thread located between the
stem (animal end) and the attachment plaque or pad
(substratum end). The strength of the entire byssus is
expected to be proportional to the number of threads
times the average strength of each thread (Bell and
Gosline, 1996). Finally, the adhesive tensile strength
is deduced from forces applied directly to byssal
plaques (Crisp et al., 1985; Young and Crisp, 1981).
Clearly, the second two strengths are subsets of mussel
tenacity and can in fact define tenacity when they become the weak link of the system. Force to break or
ultimate tensile strength measured in newtons/m2 (N/
m2) or pascals (Pa) is increasingly criticized as an inferior measure of adhesion. The energy to break is
proposed to be a better measure because it denotes the
actual work of adhesion in joules/m3. This can be determined, for example, by calculating the area under
the stress-strain curve of a sample loaded to failure.
The notion of adhesive strength persists in many biological situations because measuring extension to
break is not always feasible in field tests (see Flammang, 2002).
The most comprehensive investigation of byssal
plaque adhesion was by Crisp and his colleagues at
Menai Bridge during the 1980s (Crisp et al., 1985;
Young and Crisp, 1981). Not only did it establish that
plaque adhesion varied seasonally, but also that it was
defined by the critical surface energy of the substratum
(Table 1). Thus, weakest adhesion (about 0.15 3 105
Pa) as measured by the force or load to failure was
observed on nonpolar surfaces such as paraffin and
polytetrafluoroethylene (PTFE), whereas stronger adhesion occurred on polar surfaces e.g., glass and slate
(nearly 1 MPa). Given the range of loads to failure,
the relative magnitudes are more important than any
absolute magnitude, which on glass, for example, exhibited maxima as high as 100 times the mean load
(Young and Crisp, 1981). Significantly, Crisp et al.
(1985) were also the first to record the mode of failure
* Failure load denotes mean force for 50% failure in the samples
(n 5 146). gc represents the critical surface energy of each material
at 208C. Failure mode may denote where plaque adhesion failed:
interfacially (plaque peeling), cohesively (plaque tearing), interfacially and cohesively (mixed).
‡ Strain rate 5 0.1 sec21.
Surface
Teflon
Paraffin
Acetal
Glass
Slate
gc
mJ m22
18.5
26
40
70
100
Failure load‡
105 N m22 6 SE
0.15
0.15
1.20
7.50
3.20
8.50
5.60
6
6
6
6
1
6
1
1.08
1.00
2.57
1.11
1.12
1.01
1.15
(Jun)
(Feb)
(Jun)
(Feb)
Failure
mode
peeling
peeling
mixed
tearing
tearing
in byssal plaques. This was not a comprehensive failure analysis since chemical analysis of the fractured
surfaces was not performed. Notwithstanding this, failure on nonpolar surfaces involved primarily ‘‘peeling’’
(interfacial mode) whereas on polar surfaces plaque
tearing (cohesive failure) was common. Thus, it might
reasonably be concluded from this that mussel fouling
could be reduced by the use of materials with low
critical surface energies. Similar conclusions were
drawn from other studies (e.g., Ohkawa et al., 1999)
based on avoidance behavior of mussels on nonpolar
surfaces. In fact these observations have done little to
remedy marine fouling, because when low energy surfaces are the only ones available, mussels can compensate for the lower strength per plaque by making
more threads. Nonetheless when given a choice of substrates, the avoidance results suggest that mussels may
be able to detect different surfaces with their foot and
choose more promising polar substrata. Clearly water
as a weak boundary layer and the dielectric constant
of aqueous media do not prevent byssal adhesion on
any surface tested, but it remains to be shown how
these are specifically mitigated.
Bond strengths of synthetic industrial and specialty
adhesives often far exceed those listed in Table 1, e.g.,
100 MPa for epoxies in carbon fiber composites (Kinloch, 1987). With a level ‘‘playing field,’’ however, in
which all adhesives are applied to wet surfaces and
cured underwater, the values in Table 1 remain impressive. A recent technical bulletin from the US Department of Defense (1996) soliciting proposals on underwater adhesive technology described its goal as the
development of adhesives with modest tensile
strengths ranging from 0.2 MPa to 0.7 MPa following
a 1 hr set time. This is well within the range of mussel
adhesive strength and many other marine adhesives.
PROTEIN DIVERSITY IN ADHESIVE PLAQUES
The search for molecules responsible for byssal adhesion began with Brown’s (1952) landmark paper on
Mytilus byssus. Here, she first alluded to the peculiar
occurrence of 3, 4-dihydroxyphenylalanine- (DOPA)
containing proteins in byssus. It took nearly 30 years
1174
J. H. WAITE
TABLE 2.
The protein diversity of byssal adhesive plaques of Mytilus edulis.*
Protein
Mass (kD)
pI
mefp-1
mefp-2
mefp-3
mefp-4
mefp-5
preCol-D
110
40
6
80
9
240
.10
;9
.11
?
;9
10.7
% DOPA
13
3
20
4
30
,0.1
Repeat (n)
AKPSYPPTYK (80)
EGF motif (11)
R/NRY (4)
?
YK (8)
Gly-X-Y (175)
PTM
4-Hyp DiHyp
Cystine
Arg-OH
?
p-Ser
4-Hyp
Ref
a, b
c, d
e, f
g
h
i
* Mefp denotes M. edulis foot protein. % DOPA is in mole %. Repeat refers to recurring internal sequences with repeat frequency (n). PTM
denotes posttranslational modifications other than DOPA. These include trans-4-hydroxyproline (4-Hyp), trans-2, 3-cis-3, 4-dihydroxyproline
(DiHyp), 4-hydroxyarginine (Arg-OH), and O-phosphoserine (p-Ser).
a Waite et al. (1985); b Taylor et al. (1994a); c Inoue et al. (1995); d Rzepecki et al. (1992); e Papov et al. (1995); f Inoue et al. (1996);
g Weaver (1998); h Waite and Qin (2001); i Qin et al. (1997).
for further progress. Possibly all the precursor proteins
in the plaques are DOPA containing. The six most
abundant known plaque proteins (Table 2) have DOPA
contents ranging from 0.1 mol % in preCol-D (a byssal
collagen) to 30 mol % in mefp-5. Despite the common
presence of DOPA, the primary sequences of plaque
proteins are quite different. All exhibit repeated sequence motifs that can be long or short. Mefp-2 has
eleven repeats of the longest motif, an epidermal
growth factor (EGF) domain about 45 residues in
length (Inoue et al., 1995). In mefp-1 there are about
eighty repeats of a decapeptide sequence in which the
prolines (3 in 10) are modified to trans-4-hydroxyproline and trans-2, 3-cis-3, 4-dihydroxyproline (Laursen,
1992; Taylor et al., 1994). PreCol-D has 175 Gly-XY repeats in its collagen domain and six polyalanine
clusters in the flanking silk-like domains (Qin et al.,
1997). The shortest recurrent sequences, Arg/Arg-AsnTyr and Tyr-Lys occur in mefp-3 and -5, respectively
(Papov et al., 1995; Waite and Qin, 2001). Most of
the Arg in mefp-3 is modified to 4-hydroxyarginine
(Papov et al., 1995).
The molecular diversity of the adhesive plaques
complicates assignation of adhesion to any particular
protein(s). A common method for localizing molecules
in vivo is immunohistochemistry. This is based on the
well-known fact that the high affinity and specificity
of antibody-antigen recognition can be used to reveal
the distribution of an antigen such as a protein in tissue
thin sections. The interpretation of immunohistochemically stained plaque sections has been difficult.
There are at least two reasons for this: 1) Some of the
proteins (mefp-1, preCol-D, mefp-3) are very poor antigens (this is apparent from many failed attempts to
raise specific polyclonal antisera), and 2) the chemical
maturation of byssus may involve significant removal
or shielding of precursor epitopes. Perhaps the first
problem will be technically rectified by the use of nanoparticulate diamond powder such as that reported for
mefp-1 (Kossovsky et al., 1995). The second remains
a serious limitation that was particularly well illustrated by Anderson and Waite (2000). Specific high titer
polyclonal antibodies were raised to recombinant dpfp1, a protein precursor of Dreissena polymorpha byssal
threads. Using confocal microscopy, the antibodies
were shown to bind strongly to nascent thread material
in the foot groove. However, no binding was observed
in mature threads or proteins extracted from mature
threads. Clearly other technologies must be developed
to identify the functional distribution of proteins in
plaques.
Four years ago, we began exploring the potential of
using matrix-assisted laser desorption/ionization mass
spectrometry with time-of-flight (MALDI-TOF) to this
end. When a natural consortium of proteins is pickled
using a matrix such as sinapinic acid, then dried and
subjected to high vacuum laser irradiation, proteins in
the neighborhood of laser-excited matrix are desorbed
and ionized (Floriolli et al., 2000). Given that the penetration of the UV laser does not go deeper than 200–
300 nm into porous organic membranes (Strupat et al.,
1994), MALDI MS may be able to provide useful information about proteins near the plaque/substratum
interface. With an infrared laser this depth could be
increased ten-fold. Accordingly, plaque prints soaked
in matrix and dried were subjected to MALDI so that
the desorbed proteins could be detected according to
their mass/charge ratio (Floriolli et al., 2000). Mefp-3
and -5 were desorbed from the plaque prints of Mytilus
edulis and M. galloprovincialis byssus (Fig. 1; Floriolli et al., 2000; Warner and Waite, 1999). While
such data may seem to confirm that fp-3 and fp-5 are
present at or near the interface of the plaque and adherend, there are limits to the interpretation given the
peculiarities of MALDI TOF. For example, other proteins may be present but fail to desorb due to crosslinking, Proteins may not ionize easily, or their interaction with matrix may be suboptimal. Numerous factors contribute to protein desorption and ionization.
Suffice it say that mefp-3 is detected in plaque prints
from all tested surfaces. Given that twenty or more
mefp-3 variants may be present in the foot of an individual mussel (Warner and Waite, 1999), we were
intrigued whether the deposition of certain variants
was especially tailored to particular surfaces such as
glass, polyethylene, acrylic and steel. Although each
mussel has a different preference for the suite of mefp3 variants used in attachment, they apparently do not
deposit the variants in a surface specific manner (Floriolli et al., 2000). It is still possible, however, that
posttranslational modification of mefp-3 variants is adjusted in a surface-dependent manner. This is under
MUSSEL ADHESION
FIG. 1.
1175
MALDI TOF spectrometric analysis of a plaque ‘‘footprint’’ deposited onto stainless steel. The matrix was sinapinic acid.
active investigation. Mefp-5, in contrast, is only abundant in prints from stainless steel (Fig. 1). Taken at
face value these data suggest that mussels may be able
to tailor adhesive chemistry to surface type.
PROTEIN ADSORPTION AS A MEASURE OF ADHESION
Given that a mussel’s ability to attach to a variety
of surfaces underwater begs imitation, an inspired
biomimetic line of research would require knowing
which proteins and modifications contribute directly to
adhesion and how. This depends on a straightforward
and relevant assay for measuring adhesion of proteins.
There are many methods for testing the strength of
adhesive bonds. The following conditions are necessary for meaningful testing of byssal adhesive proteins: 1) Materials to be joined should be bonded and
loaded in water or seawater as appropriate, 2) The
method should be able to test nanomolar amounts of
protein, and 3) the method should offer high sensitivity
and fine control of force and strain rate. There are only
three techniques that satisfy all conditions: atomic
force microscopy (AFM), surface force balance, and
optical tweezers. To date only AFM has been specifically applied to mussel adhesive proteins, but not in
‘‘force’’ mode, which remains an important goal. Instead, the proteins have been evaluated as thin films
using the scanning, tapping or indentation mode of the
AFM (Baty et al., 1997; Hansen et al., 1998).
Probably because of its commercial availability
(Cell-Taky from Becton-Dickinson Bioscience, Bedford, Mass; MAP from Biopolymer AB, Sweden),
most studies of mefp adsorption and film formation
have employed mefp-1. Although mefp-1 appears to
function as a sealant or cuticle in the byssus (Miki et
al., 1996; Rzepecki et al., 1992), its basic isoelectric
point, flexible conformation (Deacon et al., 1998), and
high DOPA content (15–20 mol %) are features shared
with mefp-3 and -5, two proteins implicated in plaque
adhesion (Fig. 2). Mefp-1 adsorption has been investigated by quartz crystal microbalance (QCM), surface
plasmon resonance (SPR), ellipsometry, attenuated total reflection Fourier transform infrared spectroscopy
(ATR-FTIR), X-ray photoelectron spectroscopy
(XPS), and surface enhanced Raman spectroscopy
(SERS). SERS suggests that mefp-1 peptides chemisorb to gold and that DOPA functionalities are coordinated in edgewise fashion with the phenoxy groups
toward the gold surface (Ooka and Garrell, 2000). This
interesting observation coincides with conclusions in
an earlier study using a germanium surface (Olivieri
et al., 1989), but requires more detailed structural analyses to be conclusive. Edgewise adsorption is consistent with the thin-layer electrochemical studies of
polyphenols chemisorbed to platinum (Soriaga and
Hubbard, 1983) and various spectroscopic analyses of
pyrocatechol chemisorbed to titanium oxide (Rodriguez et al., 1996), iron and manganese oxides (McBride, 1986; McBride and Wesselink, 1988) and aluminum oxides (Kummert and Stumm, 1980; McBride
and Wesselink, 1988). Stability constants of organic
chelates on mineral or metal surfaces are thought to
approximate levels measured by solution chemistry
1176
J. H. WAITE
FIG. 2. The primary sequence of three plaque proteins that appear
to play important roles in adhesion: mefp-1, mefp-3, and mefp-5.
Highlighted amino acids are those residues in the proteins that are
targeted for modification. P is trans-4-hydroxyproline, P is trans-2,
3-cis 3, 4-dihydroxyproline, Y is Dopa, R is 4-hydroxyarginine, and
S is O-phosphoserine. See Table 2 for references.
(Kummert and Stumm, 1980). Solution studies of iron
(III)-binding by DOPA groups in mefp-1 suggest stability constants in excess of 1040 at 0.1 M NaCl, pH
7.5 and 258C (Taylor et al., 1994b, 1996). Similar affinities are predicted for pyrocatechol binding of Al
(III), Si (IV), and Ti (IV) with somewhat lower values
for transition metals (see Table 2 in Waite, 1990b). If
the DOPA functionalities of mefp-1 are indeed mobilized to chelate metals/metal oxides during chemisorption to steel, for instance, it follows that adsorbed
mefp-1 should retard corrosion. This has indeed been
demonstrated (Hansen et al., 1995).
Mefp-1 adsorption to nonpolar surfaces has been investigated using methyl-terminated alkylthiolated gold
and poly-(octadecyl methacrylate); adsorption is
patchy and disorganized (Baty et al., 1997; Höök et
al., 2001). Thin layers could be delaminated by low
shear using AFM in tapping mode, and drying led to
extensive patch perturbation (Baty et al., 1997). Protein adsorbed to polystyrene and pyrolyzed graphite
surfaces, however, showed increased durability and resistance to displacement, which was attributed to the
increased opportunity for noncovalent p-p and p-cation interactions (Baty et al., 1996; Hansen et al.,
1998). QCM analysis in parallel with SPR has great
diagnostic value because, while SPR detects the
amount of protein present in a film, QCM is also sensitive to the amount of solvent trapped or entrained in
that film (Höök et al., 2001). SPR and QCM measurement of mefp-1 adsorption to a nonpolar surface
in flow cells (Fant et al., 2000) have determined that
adsorbed layers on nonpolar surfaces are thicker, include more entrained water, and reflect a higher dissipative component (due to viscous effects) than those
adsorbed to silica. QCM and SPR both indicate that
on silica mefp-1 is adsorbed in dense rigid layers.
Comparative Langmuirian behavior of mefp-1 and -2
was investigated by FTIR on germanium and nonpolar
surfaces (Suci and Geesey, 2001). Both proteins have
similar surface coverage and adsorption rates indicating, at least on these materials, that the ten fold difference in DOPA content did not offer any special advantages.
It is important to take into account the oxidative
instability of DOPA when analyzing films made from
adsorbed mefp-1. Indeed studies of the chemical maturation of mefps provide some of the most exciting
insights into biofilms. Periodate or mushroom tyrosinase treatment of adsorbed mefp-1 both of which oxidize peptidyl-DOPA to peptidyl-DOPA-quinone,
leads to drastic physical changes in film properties.
Films become increasingly difficult to dislodge by
AFM in tapping mode (Hansen et al., 1998); also,
films shrink to roughly half their initial thickness (e.g.,
15 nm to 7 nm) and become markedly stiffer (Höök
et al., 2001). Such changes are definitely consistent
with in situ polymerization or cross-linking.
Details pertaining to the cross-linking chemistry of
DOPA proteins continue to be debated. Two covalent
pathways are currently entertained: 1) Michael-addition between DOPA-quinone and alkylamines such as
lysine, and 2) free radical aryl coupling similar to that
occurring in dityrosine or lignin formation. While the
first pathway had much popular support in the latter
half of the last century (Pryor, 1962; Laursen, 1992)
quinone alkylamine adducts have been characterized
in only a few proteins, chiefly lysyl oxidases (Wang
et al., 1996). Related quinone-imidazole adducts have
been isolated from insect cuticle (Xu et al., 1997).
Support for aryl coupling, in byssus and other DOPAcontaining structures, is growing. McDowell et al.
(1999) detected aryl-coupled products resembling
diDOPA (Fig. 3) in flow stressed byssal plaques using
solid-state 13C-NMR. Mass spectrometric analysis of
fp-1 derived decapeptides, oxidized in vitro by periodate or tyrosinase, formed multimers with masses that
were consistent with diDOPA and diDOPA-quinone
cross-link formation (Burzio and Waite, 2000). Addition of low molecular weight DOPA analogs, i.e., catechols, to the reaction medium during oxidation prevented peptide multimer formation. Instead, peptide
mass was seen to increase by the addition of one to
six catechols (Burzio and Waite, 2000). In contrast,
addition of lysine, glycine or tyrosine did little to perturb or prevent multimer formation by oxidized decapeptides.
In a recent investigation of mefp-1 adsorption to
polyvinylalcohol surfaces using SPR coupled with
light scattering, Haemers et al. (2001) reported that a
pH-dependent protein aggregation preceded adsorption. For example, average mefp-1 aggregate diameters
increased in steps from 50 nm (monomer) to 140 nm
and then to 200 nm at pH values 3.5, 4.5 and 6.5.
Aggregation was attributed to charge transfer interactions between DOPA and DOPA-quinone, but diDOPA formation can not be ruled out. FTIR analyses
of mefp-1 films oxidized by periodate or tyrosinase
MUSSEL ADHESION
1177
FIG. 3. Proposed pathway of peptidyl-DOPA-based cross-linking in plaque precursor proteins (adapted from Burzio and Waite, 2000). In step
1, dopa is converted to dopa-orthoquinone by catalysis or auto-oxidation. Dopa-quinone reacts with dopa by a reverse dismutation process (2)
to form two semiquinone free radicals. The free radicals couple through a ring- ring c-c bond (3). McDowell et al. (1999) have proposed a
5, 59 coupling based on solid-state 13C NMR analysis of plaques. Di-dopa can easily be reoxidized once or twice to a diquinone (4). Such
oxidation steps are suggested by mass spectrometry (Burzio and Waite, 2000). Involvement of lysine in Schiff-base additions does not drive
peptide cross-linking, but may occur after di-dopa cross-links have been formed (5). This is based on losses of lysine in oxidized peptides
(Burzio and Waite, 2000).
generally concurred with the absence of quinone-alkylamine adducts, but it also exposed a possible new
complication: amino groups in mefp-1 and polylysine
appear to be unstable to periodate treatment (Suci and
Geesey, 2000). Both polymers show NH2 losses apparently associated with periodate treatment. The
chemistry involved and significance, if any, to crosslinking in byssal adhesive plaques remain unknown.
Biomimetic attempts. Availability of the complete
primary sequence of several plaque-derived proteins
has inspired attempts to produce complete or partial
biomimetic analogs. All efforts to date pertain to mefp1 and its tandemly repeated decapeptide (Fig. 2). The
field abounds with reports of the synthesis of ‘‘approximate’’ DOPA- and hydroxyproline-containing
decapeptides (see Olivieri et al., 1989; Swerdloff et
1178
J. H. WAITE
al., 1989), however, Yamamoto (1987) significantly
advanced the field by using a fragment condensation
strategy to make a polymer with ten decapeptide repeats (10mer). A more fastidious synthesis of the decapeptide replete with DOPA and hydroxyproline and
dihydroxyproline has been reported (Taylor and Weir,
2000). A comparison of the conformation and surface
binding properties of this decapeptide with native and
more approximate synthetic versions should prove interesting as two conflicting conformations have been
proposed for synthetic decapeptides without dihydroxyproline: a bent right-handed a-helix (Olivieri et al.,
1997) and a left-handed type II polyproline helix (Kanyalkar et al., 2002). Sufficient quantity of the 10mer
was produced to attempt some tests of tensile strength.
On steel coupons, however, this analog showed bonding strengths (;2.8 MPa) that were less than half those
of poly-L-lysine and gelatin on steel at 60% humidity
(Yamamoto, 1987). It is not clear from the report
whether a cross-linking catalyst was included or what
the pH of the polymer solution was. Furthermore, expectations of ‘‘heroic’’ adhesive tensile strength may
not be realistic under any conditions given that native
mefp-1 functions as a protective sealant in byssus.
Simpler adhesive analogs of the mefps have been
prepared as random copolymers of DOPA and lysine
or glutamic acid (Yu and Deming, 1998; Yu et al.,
1999). These have provided some penetrating insights
into the possible chemical role of DOPA in adhesion.
Regarding involvement of lysine in cross-link formation, random copolymers of DOPA (20 mol %) and
lysine were compared with those of DOPA (20 mol
%) and glutamic acid in lap shear adhesion on aluminum substrates. There was no significant difference
in the performance of these as adhesives (;5 MPa).
Regarding the proportion of DOPA, random copolymers were prepared with three different DOPA concentrations (0, 10 and 20 mol %): adhesive bonding
strength increased directly with DOPA concentration.
Finally, with respect to chemical maturation, it was
demonstrated using the random copolymers (DOPA:
Lys ratio 1:4) that optimal adhesion depended on limited polymer oxidation. Using hydrogen peroxide as
the DOPA oxidant, Yu et al. (1999) showed that absence of oxidant led to cohesive failure in the adhesive, while high oxidant levels (molar ratio of DOPA:
H2O2 5 1:0.25) resulted in interfacial failure. Highest
lap shear bond strengths (5 MPa) were achieved at
intermediate levels of oxidation (1:0.05). These data
support what has already been suggested: that the
DOPA side-chain in proteins serves two important
roles in adhesion—the formation of cross-links and
interfacial complexes in chemisorption (Waite et al.,
1992). Exactly how mussels balance these mutually
competing needs remains to be further investigated. It
should be pointed out that adhesive bonding tests using
biomimetic polymers have yet to assemble and test
their bonded materials underwater.
CONCLUSIONS
The permanent and opportunistic byssal attachment
of mussels is derived from an assortment of proteins
that show extensive posttranslational modifications.
Chief among these is DOPA, which occurs at highest
levels in those proteins distributed near the plaquesubstratum interface. In an attempt to understand the
role of DOPA-proteins in events leading up to adhesion, many researchers have studied the adsorption of
DOPA-peptides and proteins to surfaces. Those features of DOPA protein adsorption with which most
researchers concur are itemized as follows: 1) mefp-1
and -2 adsorb rapidly and irreversibly to various surfaces; 2) the morphologies of adsorbed mefps are surface dependent; 3) the mechanical properties of adsorbed mefps are surface dependent; 3) the irreversibility of adsorption may be related to two features: on
polar surfaces mefps are likely to be chemisorbed by
reactive functionalities, e.g., phospho-esters and
DOPA. On all surfaces including nonpolar types, irreversibility of adsorption is enhanced by protein aggregation. Because aggregation is driven by increasing
pH or periodate oxidation, it is believed to involve
charge transfer between DOPA and DOPA-quinone or
aryl coupling (i.e., diDOPA); 4) Given their strong net
positive charge mefps may be predisposed to coadsorption with polyanions (Höök et al., 2001; Suci and
Geesey, 2000).
DOPA-based adhesive strategies are probably fairly
rare in nature having been positively detected only in
marine and freshwater mussels, tunicates, reef-building
polychaetes, and some trematodes (Waite, 1990a).
Notwithstanding this, it is proposed that a deeper understanding of the molecular mechanism of byssal adhesion would lead to fundamental insights with regard
to adhesion in nature.
ACKNOWLEDGMENTS
I thank Peter Suci, Manoj Chaudhuri, Fredrik Höök
and Hans Elwing, for sharing insightful speculations
about adhesion and protein adsorption.
REFERENCES
Anderson, K. E. and J. H. Waite. 2000. Immunolocalization of
Dpfp1, a byssal protein of the zebra mussel (Dreissena polymorpha). J. Exp. Biol. 203:3065–3076.
Aristoteles. 1910. Historia animalium. Vol. 4. Trans. Thompson,
d’Arcy W. Oxford University Press, Oxford.
Baty, A. M., P. K. Leavitt, C. A. Siedlecki, B. J. Tyler, P. A. Suci,
R. E. Marchant, and G. G. Geesey. 1997. Adsorption of adhesive proteins from the marine mussel, Mytilus edulis, on polymer films in the hydrated state using angle dependent X-ray
photoelectron spectroscopy and AFM. Langmuir 177:307–315.
Baty, A. M., P. A. Suci, B. J. Tyler, and G. G. Geesey. 1996. Investigation of mussel adhesive protein adsorption on polystyrene and poly (octyl methacrylate) using angle dependent XPS,
ATR-FTIR, and AFM. J. Colloid & Interf. Sci. 177:307–315.
Bell, E. C. and J. M. Gosline. 1996. Mechanical design of mussel
byssus: Material yield enhances attachment strength. J. Exp.
Biol. 159:197–208.
Bell, E. C. and J. M. Gosline. 1997. Strategies for life in flow:
Tenacity, morphometry, and probability of dislodgment of two
Mytilus species. Marine Ecology Progress Series 159:197–208.
MUSSEL ADHESION
Brown, C. H. 1952. Some structural proteins of Mytilus edulis, L.
Q. J. Microsc. Sci. 93:487–502.
Burzio, L. A. and J. H. Waite. 2000. Cross-linking in adhesive quinoproteins: Studies with model decapeptides. Biochemistry 39:
11147–11153.
Cayless, R. A. 1991. Interfacial chemistry and adhesion: The role
of surface analysis in the design of strong stable interfaces for
improved adhesion. Sur. Inter. Anal. 17:430–438.
Crisp, D. J., G. Walker, G. A. Young, and A. B. Yule. 1985. Adhesion and substrate choice in mussels and barnacles. J. Coll.
Interf. Sci. 104:40–50.
Deacon, M. P., S. S. Davis, J. H. Waite, and S. E. Harding. 1998.
Structure and mucoadhesion properties of mussel glue protein
in dilute solution. Biochemistry 37:14108–14112.
Fant, C., K. Sott, H. Elwing, and F. Höök. 2000. Adsorption behavior
and enzymatically or chemically induced cross-linking of a
mussel adhesive protein. Biofouling 16:119–132.
Flammang, P., J. Ribesse, and M. Jangoux. 2002. Biomechanics of
adhesion in sea cucumber cuvierian tubules (Echinodermata,
Holothuroidea). Integr. Comp. Biol. 42:1107–1115.
Floriolli, R. Y., J. von Langen, and J. H. Waite. 2000. Marine surfaces and the expression of specific byssal adhesive protein variants in Mytilus. Mar. Biotech. 2:352–363.
Haemers, S., M. C. van der Leeden, E. J. Nijman, and G. Frens.
2001. The degree of aggregation in solution controls the adsorbed amount of mussel adhesive proteins on a hydrophilic
surface. Coll. Surf. 190:193–203.
Hansen, D. C., S. C. Dexter, and J. H. Waite. 1995. The inhibition
of corrosion of S30403 stainless steel by a naturally occurring
catecholic polymer. Corrosion Sci. 37:1423–1441.
Hansen, D. C., S. G. Corcoran, and J. H. Waite. 1998. Enzymatic
tempering of a mussel adhesive protein film. Langmuir 14:
1139–1147.
Holubka, J. W., J. E. deVries, and R. A. Dickie. 1984. Interfacial
chemistry of humidity induced adhesion loss. Ind. Eng. Chem.
Prod. Res. Dev. 23:63–70.
Höök, F., B. Kasemo, T. Nylander, C. Fant, K. Sott, and H. Elwing.
2001. Variations in coupled water, viscoelastic properties and
film thickness of a mefp-1 protein film during adsorption and
cross-linking: A quartz crystal microbalance with dissipation
monitoring, ellipsometry and surface plasmon resonance study.
Anal. Chem. 74:5796–5804.
Inoue, K., Y. Takeuchi, D. Miki, and S. Odo. 1995. Mussel adhesive
plaque protein gene is a novel member of epidermal growth
factor-like gene family. J. Biol. Chem. 270:6698–6701.
Inoue, K., Y. Takeuchi, D. Miki, S. Odo, S. Harayama, and J. H.
Waite. 1996. Cloning, sequencing, and sites of expression for
the hydroxyarginine-containing plaque protein of the mussel
Mytilus galloprovincialis. Eur. J. Biochem. 239:172–176.
Israelachvili, J. N. 1985. Intermolecular and surface forces with applications to colloidal and biological system. Academic Press
Inc. Ltd., London 12–51.
Kanyalkar, M., S. Srivastava, and E. Coutinho. 2002. Conformation
of a model peptide of the tandem repeat decapeptide in mussel
adhesive protein by NMR and MD simulations. Biomaterials
23:389–396.
Kinloch, A. J. 1987. Adhesion and adhesives. Science and technology. Chapman and Hall, London.
Kossovsky, N., A. Gelman, H. J. Hnatyszyn, S. Rajguru, R. L. Garrell, S. Torbati, S. F. Freitas, and G. M. Chow. 1995. Surface
modified diamond nanoparticles as antigen delivery vehicles.
Bioconjugate Chem. 6:507–511.
Kummert, R. and W. Stumm. 1980. The surface complexation of
organic acids on hydrous g-alumina. J. Colloid Interf. Sci. 75:
373–385.
Laursen, R. 1992. Reflections on the structure of mussel adhesive
proteins. Results Probl. Cell Differentiation 19:52–72.
McBride, M. B. 1986. Adsorption and oxidation of phenolic compounds by iron and manganese oxides. Soil Sci. Soc. Am. J.
51:1466–1472.
McBride, M. B. and L. G. Wesselink. 1988. Chemisorption of catechol on gibbsite, boehmite, and noncrystalline alumina. Environ. Sci. Technol. 22:703–708.
1179
McDowell, L. M., L. A. Burzio, J. H. Waite, and J. Schaefer. 1999.
REDOR Detection of cross-links formed in mussel byssus under
high flow stress. J. Biol. Chem. 274:20293–20295.
Miki, D., Y. Takeuchi, K. Inoue, and S. Odo. 1996. Expression sites
of two byssal protein genes of Mytilus galloprovincialis. Biol.
Bull. 190:213–217.
Ohkawa, K., A. Nishida, R. Honma, Y. Matsui, K. Nagaya, A. Yuasa, and H. Yamamoto. 1999. Studies on fouling by the freshwater mussel Limnoperna fortunei and the antifouling effects of
low energy surfaces. Biofouling 13:337–350.
Olivieri, M. P., R. E. Baier, and R. E. Loomis. 1989. Surface properties of mussel adhesive protein component films. Biomaterials
13:1000–1008.
Olivieri, M. P., R. M. Wollman, and J. L. Alderer. 1997. Nuclear
magnetic resonance spectroscopy of mussel adhesive protein repeating peptide segment. J. Pept. Res. 50:436–442.
Ooka, A. A. and R. L. Garrell. 2000. Surface enhanced Raman spectroscopy study of DOPA-containing peptides related the adhesive protein of the marine mussel Mytilus edulis. Biopolymers
57:92–102.
Papov, V. V., T. V. Diamond, K. Biemann, and J. H. Waite. 1995.
Hydroxyarginine-containing polyphenolic proteins in the adhesive plaques of the marine mussel, Mytilus edulis. J. Biol.
Chem. 270:20183–20192.
Price, H. A. 1983. Structure and formation of the byssus complex
in Mytilus (Mollusca, Bivalvia). J. Moll. Stud. 49:9–17.
Pryor, M. G. M. 1962. Sclerotization. Comp. Biochem. 4:371–396.
Qin. X. X., K. J. Coyne, and J. H. Waite. 1997. Tough tendons:
Mussel byssus has collagen with silk-like domains. J. Biol.
Chem. 272:32623–32627.
Rodriguez, R., M. A. Blesa, and A. E. Regazzoni. 1996. Surface
complexation of the TiO2 (anatase)/aqueous solution interface
of catechol. J. Coll. Interf. Sci. 177:122–131.
Rzepecki, L. M., K. M. Hansen, and J. H. Waite. 1992. Characterization of a cystine rich polyphenolic protein from the blue
mussel Mytilus edulis L. Biol. Bull. 183:123–137.
Schonhorn, H. 1981. Adhesion and adhesives: Interactions at interfaces. In J. F. Oliver, (ed.), Adhesion in cellulosic and woodbased composites, pp. 91–111. Plenum, New York.
Soriaga, M. and A. T. Hubbard. 1983. Electrochemistry of chemisorbed molecules: Effect of chirality on the orientation and electrochemical oxidation of l and dl-DOPA. J. Phys. Chem. 87:
232–235.
Strupat, K., M. Karas, F. Hillenkamp, C. Eckerskorn, and F. Lottspeich. 1994. Matrix-assisted laser desorption ionization mass
spectrometry of proteins after polyacrylamide gel electrophoresis. Anal. Chem. 66:464–470.
Suci, P. A. and G. G. Geesey. 2000. Influence of sodium periodate
and tyrosinase on binding of alginate to adlayers of Mytlius
edulis foot protein 1. J. Colloid Interf. Sci. 230:340–348.
Suci, P. A. and G. G. Geesey. 2001. Comparison of adsorption behavior of two Mytilus edulis foot proteins on three surfaces.
Coll. Sur. 22B:159–168.
Swerdloff, M. D., S. B. Anderson, R. D. Sedgwick, M. K. Gabriel,
R. J. Brambilla, D. M. Hindenlang, and J. I. Williams. 1989.
Solid-phase synthesis of bioadhesive analogue peptides with trifluoromethanesulfonic acid cleavage from PAM. Int. J. Peptide
Protein Res. 33:318–327.
Taylor, C. M. and C. A. Weir. 2000. Synthesis of the repeating decapeptide unit of Mefp1 in orthogonally protected form. J. Org.
Chem. 65:1414–1421.
Taylor, S. W., J. H. Waite, M. M. Ross, J. Shabanowitz, and D. F.
Hunt. 1994a. Trans-2, 3-cis-3, 4-dihydroxyproline, a new naturally occurring amino acid, is the sixth residue in the tandemly
repeated consensus decapeptides of an adhesive protein from
Mytilus edulis. J. Amer. Chem. Soc. 116:10803–10804.
Taylor, S. W., G. W. Luther, and J. H. Waite. 1994b. Polarographic
and spectrophoto-metric investigation of Fe (III) complexation
to 3, 4-dihydroxyphenylalanine-containing peptides and proteins from Mytilus edulis. Inorg. Chem. 33:5819–5824.
Taylor, S. W., D. B. Chase, M. H. Emptage, M. J. Nelson, and J. H.
Waite. 1996. Ferric ion complexes of a Dopa-containing adhesive protein from Mytilus edulis. Inorg. Chem. 35:7572–7577.
1180
J. H. WAITE
United States Department of Defense. 1996. Underwater adhesive
requirements. Bulletin CSS/TN 96/7: 4.
Van der Feen, P. J. 1949. Byssus. Basteria 13:66–71.
Waite, J. H. 1992. Formation of mussel byssus: Anatomy of a natural
manufacturing process. Results Probl. Cell Differentiation 19:
27–54.
Waite, J. H. 1983. Adhesion in byssally attached bivalves. Biol. Rev.
58:209–231.
Waite, J. H. 1990a. The phylogeny and chemical diversity of quinone-tanned glues and varnishes. Comp. Physiol. Biochem.
97B:19–29.
Waite, J. H. 1990b. Marine adhesive proteins: Natural composite
thermosets. Int. J. Biol. Macromol. 12:139–144.
Waite, J. H., T. J. Housley, and M. L. Tanzer. 1985. Peptide repeats
in a mussel glue protein: Theme and variations. Biochem. 24:
5010–5014.
Waite, J. H., R. Jensen, and D. E. Morse. 1992. Cement precursor
proteins of the reef-building polychaete Phragmatopoma californica (Fewkes). Biochemistry 31:5733–5738.
Waite, J. H. and X. X. Qin. 2001. Polyphenolic phosphoprotein from
the adhesive pads of the common mussel. Biochemistry 40:
2887–2893.
Wang, S. X., M. Mure, K. F. Medzihradszky, A. L. Burlingame, D.
E. Brown, D. M. Dooley, A. J. Smith, H. K. Kagan, and J. P.
Klinman. 1996. A cross-linked cofactor in lysyl oxidase: Redox
function for amino acid side chains. Science 273:1078–1082.
Warner, S. C. and J. H. Waite. 1999. Expression of multiple forms
of an adhesive plaque protein in an individual mussel, Mytilus
edulis. Mar. Biol. 134:729–734.
Weaver, J. L. 1998. Isolation, purification and partial characterization
of a mussel byssal precursor protein, Mytilus edulis foot protein
4. Master’s Thesis, University of Delaware.
Xu, R., X. Huang, T. L. Hopkins, and K. J. Kramer. 1997. Catecholamine and histidyl protein cross-linked structures in sclerotized insect cuticle. Insect Biochem. Molec. Biol. 27:101–108.
Yamamoto, H. 1987. Synthesis and adhesive studies of marine polypeptides. J. Chem. Soc. Perkin Trans. I 1987:613–618.
Young, G. A. and D. J. Crisp. 1982. Marine animals and adhesion.
In K. W. Allen (ed.), Adhesion 6. Barking, Applied Science
Publishers, Ltd, England 19–39.
Yu, M. and T. J. Deming. 1998. Synthetic polypeptide mimics of
marine adhesives. Macromolecules 31:4739–4745.
Yu, M., J. Hwang, and T. J. Deming. 1999. Role of l-3, 4-dihydroxyphenylalanine in mussel adhesive proteins. J. Am. Chem. Soc.
121:5825–5826.