To begin to address this question, a good portion of my graduate career was spent collecting mussels from the Puget Sound, bringing them into the laboratory, and encouraging them (to the best of my ability) to attach to manufactured plates. I would then cut the threads and move the plates with attached plaques into different seawater conditions (various combinations of seawater pH, dissolved oxygen, and temperature), incubating them for up to 20 days. This approach was taken in order to investigate the direct interaction between seawater and the material, without confounding our results by stressing out the animal. While the glue was incubating, I would periodically remove plates from seawater treatments and pull adhesive plaques to 'failure', using a motor-driven mechanical testing machine that was capable of measuring the force required to remove the adhesive. With this experimental design, I was able to study the natural ‘life-cycle’ of the adhesive while also isolating which aspects of the seawater environment are critical to its function.
After patiently waiting for my mussels to cooperate, I was rewarded with an answer: mussel glue functions as a two-stage epoxy that uses the pH and oxygen content of seawater as molecular triggers (Figure 4A)! Applied by the animal in a semi-fluidic state, plaque proteins initially adhere to a surface while a mussel’s foot is pressed against it and no water is present. Once all the proteins are deposited, the mussel lifts its foot and seawater comes in contact with the adhesive, drastically increasing the pH and oxygen availability around the plaque. In response, the glue begins to ‘cure’, transitioning from a fluid to a solid as L-DOPA functional groups incorporate oxygen and form crosslinks with one another, a process that is facilitated by pH-sensitive enzymes. Evidence for this transition was observed in the laboratory as the strength of the adhesive doubled over the course of 8 days in ‘typical’ seawater conditions, while the adhesive slowly turned from white to a dark tan color, indicating the formation of the crosslinked form of L-DOPA, DOPA-quinone (Figure 4B-E). This process was almost completely halted when seawater pH (<5.0) and dissolved oxygen (<2 mg L-1) were kept low, indicating that both were required to motivate the change in the material.