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