Understanding polymorph production and control in calcite/aragonite biominerals

As children we all looked forward to our beach holidays, playing in the sand, building castles. These ephemeral structures we would often decorate with shells gathered from the foreshore. Sometimes luck prevailed and we found opened shells but more than likely we would scavenge and find mussels attached to nearby rocks. In Scottish waters these were more than likely the common blue mussel, Mytilus edulis, which, should we try and open them or smash them with a rock, we would find quite resilient, tough. This resilience is due to the unique shell structure laid down by the mollusc as it grows. How it does this is by recruiting certain proteins, unique to molluscs, to convert the basic shell material, calcium carbonate, into ordered layered structures. If you look at the external surface of the mussel it is rough and with a bit of effort you may be able to dislodge some of the surface coat. However, if you look at the inside of the shell there is a pearlescent material, called nacre, which is many times tougher than the outer coat. By cutting the shell in a specific direction and using an electron microscope to look in great detail at the arrangement of this calcium carbonate we find two forms (called polymorphs) - calcite on the outer layer and aragonite in the inner (nacre) layer. Although calcite readily forms from calcium carbonate in the laboratory, aragonite is a high-pressure polymorph which, as the name suggests, requires high pressure for the tougher form to be produced. A good analogy is that aragonite is to calcite as diamond is to graphite / both materials are made from the same chemical elements, but have quite different characteristics. The purpose of this project is to determine how the humble sea mollusc produces calcite and aragonite at ambient temperature and pressure, a feat that is not possible under normal laboratory conditions. To do this we have to examine both the shell architecture and also the proteins that may be recruited to accomplish this production of calcite and aragonite. The shell architecture will be looked at in fine detail by using two techniques / scanning electron microscopy (SEM) and electron back-scatter diffraction (EBSD). This will allow us to see this arrangement of calcite and aragonite within the shell. Simultaneously, we will look at a number of proteins that are found in the extrapallial (EP) fluid / which is found between the nacre layer and the soft (edible) part of the mussel and is easily extracted with a syringe. This EP fluid, which contains a number of different proteins, is thought to be the source of proteins needed to carry out the transformation from calcite to aragonite. In this project, several of these individual proteins will be isolated from the EP fluid and used to determine exactly which ones influence this change from calcite to aragonite. SEM and EBSD will be used to follow the growth of calcite/aragonite in a laboratory environment and hence we can determine which proteins cause the switch. Within the EP fluid there is one protein that stands out more than all the others and we will investigate this protein first since this is the most likely candidate for transformation. The most exciting way to do this is to determine its 3-D structure by using X-ray diffraction, a technique that the protein group in Glasgow excels. By knowing the structure we can determine how it works. So, what good is all this? Well, two important aspects: firstly, if you can control this switching you can sequentially lay down different layers on a number of different substrates (which we will also investigate). There is evidence that the aragonite nacre could potentially be exploited in the stimulation of bone production in osteoporosis for example if synthetic nacre could be provided in a suitable form. Secondly, the physical characteristics (extreme hardness) of aragonite nacre could also be exploited in a number of ways / protection for fragile surfaces and humans.

The ability of simple marine bivalves to control calcium carbonate polymorph production forming calcite and the high-pressure polymorph, aragonite, will be investigated. Central to this control are specific proteins that catalyse nucleation and growth, inducing polymorph switching and inhibition of growth. The influence of individual extrapallial fluid (EP) proteins on number, size and crystallography of in vitro calcium carbonate crystals (Kitano protocol) will be assessed using scanning electron microscopy (SEM) electron backscatter diffraction (EBSD) to monitor the effect that pure, well characterised proteins have on crystal growth in vitro. Those EP proteins (+/- carbohydrate) that influence calcium carbonate growth will be fully characterized with the ultimate aim of determining the conditions for crystallization allowing the elucidation of their three-dimensional structures. Protein purification by ammonium sulphate cuts, ion exchange and gel filtration using AKTA FPLC. Protein crystallisation trials by robotic screening employing Hamilton microlab star & Cartesian nano-drop dispenser with protein crystals produced analysed for suitability using a Rigaku X-ray diffractometer. With pure proteins from two species of bivalve molluscs, Mytilus edulis and Modiolus modiolus, that have different shell morphology (ratio of calcite to aragonite), we shall begin to characterise those proteins that influence polymorph switching by 2-D gel electrophoresis (GE) and peptide mass fingerprinting by MALDI or ESI (University of Dundee). Nano-fabrication will employ electron beam lithography and dry etching to form nano-patterns in calcite and chitin into which these pure proteins can be introduced sequentially to assess the feasibility of the production of layered interconnected calcite-aragonite structures of predetermined shape and material properties.