The Inside Scoop (NIDCR) For most people, mussels are strictly
an “on-the-half-shell” indulgence. But if you stop to look inside
the shell, you will notice a large, round muscular structure,
or foot.
It tells a remarkable story of evolution. In nature, an adult
mussel extends this foot down into its watery habitat, probing
for a suitable resting place. Once found, the foot presses firmly
against its chosen surface, secreting first a glue-like adhesive
pad as a base of attachment. In the meantime, additional secretions
from the foot harden into elastic protein threads that tether
the mussel into place and make it utterly resistant to the surge
of the sea, the whipping of the winds, or any other sheer force
that nature can throw its way. Remarkably, mussels can adhere
to surfaces wet and dry, organic and inorganic.
This all-purpose adhesiveness first intrigued scientists a few
decades ago as one of nature’s best guides to design better dental
and medical bioadhesives. However, attempts to mimic mussels have
been slowed by an inadequate understanding of the molecular underpinnings
of their adhesion. But, as new and more powerful research technologies
have emerged, these mechanisms are starting to come into focus.
In a groundbreaking study published online this August in the
Proceedings of the National Academy of Sciences, NIDCR grantees
and a colleague defined the adhesive qualities of a single amino
acid that is prominent in mussel glue. The Inside Scoop spoke
to Dr. Phillip Messersmith, the senior author on the paper and
a scientist at Northwestern University in Evanston, IL, to hear
his perspective.
Most dentists have heard about the promise of mussel glue as
a bioadhesive. But your paper tells a different story in that
you evaluate one key component of a mussel's adhesive pad called
dopa. What is dopa? Dopa is an amino acid formally known as 3,
4-dihydroxy-L-phenylalanine. What’s fascinating about dopa is
it is not encoded in the DNA of any of the mussel families.
In fact, it’s not encoded in the DNA of any organism that I’m
aware of, including the genetic material of mammals. So how do
mussels produce dopa? From the amino acid tyrosine. Mussels have
evolved a set of enzymes that convert tyrosine into dopa. How
much dopa does a mussel produce? That’s tough to answer. You mentioned
the mussel’s adhesive pad.
We know of at least five specialized glue proteins present in
the adhesive pad of the widely studied blue mussel, Mytilus edulis.
All have high concentrations of dopa, ranging from a few to 27
mol percent. You may already know, “a mol percent” is the amount
of a given amino acid within a protein calculated as a percentage
of the total protein. The ubiquity of dopa within these proteins
has fueled speculation that it must be a critical adhesive component
of mussel glue proteins. Why speculation only? Because of the
level of evidence.
There was no molecular evidence that would allow one to say definitively
that dopa plays a significant role in adhesion. That was basically
the focus of this paper. What does an individual dopa amino acid,
or residue, contribute to adhesiveness? There’s no way to answer
that question using a whole protein. We had to isolate the individual
amino acids. What did you find? If you take a dopa at neutral
pH and touch it down on a hard titanium oxide surface, you measure
a pull-off force that is extraordinarily high - 800 piconewtons,
which translates to .8 nanonewtons. Wait a second. What's a pico-
and a nanonewton? They’re standard units of force. To put these
numbers into perspective, the strongest known chemical bond is
a covalent bond. It’s the workhorse. Using similar methodology,
a covalent bond would break at about 1 to 3 nanonewtons. So, the
interaction of dopa with an oxide surface approaches the strength
of a covalent bond.
But it’s not a covalent bond. That’s one of the unique aspects
of our finding. You have this extraordinarily high interaction
force, and yet the bond is fully reversible. And that flies in
the face of everything you learned in biochemistry 101? The paradigms
usually are: High strength bonds are irreversible, while low strength
bonds are reversible. Most biological interactions, like receptor-ligand
interactions, are on the order of 200 piconewtons. Those you can
break and reform indefinitely. But they are several times smaller
in force values. So, this interaction was very interesting in
that it seemed to be somewhere in this gap between low strength
reversible and high strength irreversible. When you got these
results, what did you think? My first thought was, “Is this real?”
Everything is so small that you can never confirm that you have
a single molecule interaction. We repeated the experiment many
times and generated some other data, and that led us to the conclusion
that, yeah, this is exactly what we thought we were measuring.
So it's very reproducible? Absolutely. Tell me, how did you isolate
the dopa residues? We used atomic force microscopy, or ATM. If
you’ve ever read about the technique, it involves measuring the
force of interaction between a very sharp tip and a surface. But,
to perform our specific experiments, we designed our ownmethodology.
How so? First, we had to make specialized AFM tips. These tips
were made of rigid, inorganic material called silicon nitride
sharpened down to a radius of approximately 10 or 20 nanometers.
We also outfitted the AFM tips with a polymer tether. The individual
dopas linked chemically to the polymer tether and, from there,
the measurements were fairly straightforward. So, to recap, we
took the tip, touched it down on the surface, then attempted to
pull off a dopa. That allowed us to measure the interaction force
necessary to debond or disengage the dopa from the surface. But
the trick was getting just one amino acid on the tip. Is that
correct? Right, that’s the hard part. When you bring the AFM tip
down on the surface, you have a roughly one in 10 chance of measuring
any dopa interaction. In other words, you get such a vanishingly
low concentration of dopa on the tip that, first of all, most
tips never work because they don’t pick up any dopa residues.
So, let’s do the math.
If you want to measure a pull off force of dopa on a surface
and get, say, 100 of these forces to generate a nice graphical
display of data, you’ve got to do that experiment 1,000 times
roughly. By the way, these technical hurdles are not specific
to dopa. It’s just the nature of any single molecule experiment.
After your initial experiments on the titanium oxide surface,
what did you study next? Well, we did the same experiment, except
at the pH of sea water and then even higher. As expected, the
dopa became oxidized and was less adhesive to the titanium oxide.
The surface adhesion dropped by seven or eight times,
which is roughly on the order of 100 piconewtons. Then you switched
to an organic surface. That’s right. We created a surface that
contained amines. They are organic compounds that often are found
on the surface of proteins. At marine pH values, we measured a
two nanonewton rupture force on our organic surface but - and
this is a key point - the bond didn’t reform. That meant we had
just broken a covalent bond on the organic surface. Why a covalent
bond? Well, it had been hypothesized previously that the chemical
reaction between an oxidized dopa and an amine would produce a
covalent bond. But what had never been demonstrated until now
is that this reaction can occur on a surface. Does the covalent
bond reform? No. Just like any other covalent bond, it can’t reform
under these conditions. But if the mussel is motile?
You bring up a good question. What if a mussel wants to move?
They can move, but generally they don’t pull off their adhesive
pads. They basically just cut the threads off and leave them behind.
They secrete a new adhesive pad. What’s interesting is it appears
that dopa has the ability to form very strong interactions to
both organic and inorganic surfaces. That’s part of the story
that I think is developing towards at least adding to the understanding
of how a mussel might be able to attach to different surfaces
in nature. These dopa residues have a versatility in adhesion
that is not seen in other amino acids. Where does your group go
from here? We’re still continuing our work in mimicking these
proteins in synthetic biomaterials. That work continues and, honestly,
benefits from a better understanding of the fundamental adhesion.
How so? We worked for several years in mimicking mussel adhesive
proteins with synthetic polymers that incorporate dopa. All of
this work was undertaken without our current level of understanding.
And we weren’t the only ones. But now we have a level of understanding
that, I think, will allow us to better design synthetic polymers
that can function as nice surgical adhesives. And how might this
finding change your thinking about synthesizing these adhesives?
Well, it has given us a lot to think about when considering adherence
to mineralized tissue, such as dental enamel, dentin, and bone.
I point this out because those tissues have high organic and inorganic
components. If you can design a molecule that has the ability
to bond to both the inorganic and organic components, I think
you stand a better chance of developing a useful adhesive than
if you have one that only targets the organic or inorganic component.
You'd have both bases covered? Right. Bone, for example, is roughly
60 percent mineral. The rest is water and protein.
That’s actually a reasonably good balance, and you’d like to
be able to adhere to both of those components. So the trick is
to learn how to build that versatility into your adhesive? Yeah.
Our experiments also give us some ideas about how to do that.
Control of pH is one of the key elements. Where do you see this
research benefitting patients? Even though we set out to make
adhesive polymers for tissue adhesion, I think one of our really
exciting discoveries is dopa containing polymers can prevent adhesion
and fouling of surfaces. I think this is the most compelling near-term
application for this research. The idea is conceptually simple.
You use dopa or a peptide containing dopa as a sticky anchor to
adhere an anti-fouling polymer onto a surface. Envision, for example,
a medical device, such as a catheter or a vascular stent. Or,
in the oral cavity, think of a denture. All can become fouled
in one way or another over time, with proteins, cells, bacteria,
or yeast, and that colonization is problematic for their performance.
We’ve developed a way to exploit the adhesiveness of dopa to adhere
a polymer down on the surface.
The anchored polymer serves as a repellant for subsequent bioadhesion.
It’s sort of an ironic twist to all of this, given that we wanted
to make things adhesive. But we were inspired by one of nature’s
best fouling organisms. Right, there are two sides to every story.
That’s right. My strong conviction is that the anti-fouling coatings
will be the first practical outcome of our work. What about better
adhesives in dentistry? We’re working on that. We think