Understanding polymorph production and control in calcite/aragonite biominerals

作为孩子，我们都期待着我们的海滩假期，在沙滩上玩耍，建造城堡。我们常常用从海边采来的贝壳来装饰这些短暂的建筑物。有时运气占上风，我们会发现打开的贝壳，但更有可能的是，我们会捡到附着在附近岩石上的贻贝。在苏格兰的海水中，这些很可能是常见的蓝贻贝，即紫贻贝，如果我们试着打开它们，或者用石头砸它们，我们会发现它们很有弹性，很坚硬。这种弹性是由于软体动物生长过程中形成的独特外壳结构。它如何做到这一点，是通过招募软体动物特有的某些蛋白质，将基本的外壳材料碳酸钙转化为有序的层状结构。如果你看贻贝的外部表面，它是粗糙的，用一点努力，你可能能够去除一些表面的外套。然而，如果你看一下贝壳的内部，有一种珠光物质，叫做珠层，它比外壳坚硬很多倍。通过在一个特定的方向切割外壳，并使用电子显微镜仔细观察这种碳酸钙的排列，我们发现了两种形式（称为多晶型）——外层的方解石和内层的文石（珍珠层）。虽然方解石很容易在实验室中由碳酸钙形成，文石是一种高压多晶体，顾名思义，需要高压才能产生更坚硬的形式。一个很好的类比是文石之于方解石，就像金刚石之于石墨/这两种材料是由相同的化学元素构成的，但具有完全不同的特征。这个项目的目的是确定卑微的海洋软体动物如何在环境温度和压力下产生方解石和文石，这在正常的实验室条件下是不可能的。为了做到这一点，我们必须检查外壳结构和蛋白质，这些蛋白质可能被招募来完成方解石和文石的生产。壳结构将通过使用扫描电子显微镜（SEM）和电子背散射衍射（EBSD）两种技术来详细研究。这将使我们看到方解石和文石在壳内的排列。同时，我们将研究在贻贝的珍珠层和软（可食用）部分之间的表皮外液（EP）中发现的一些蛋白质，这些蛋白质很容易用注射器提取。这种EP流体含有许多不同的蛋白质，被认为是方解石向文石转化所需蛋白质的来源。在这个项目中，将从EP液中分离出这些单独的蛋白质，并用于确定哪些蛋白质影响方解石到文石的变化。SEM和EBSD将用于在实验室环境中跟踪方解石/文石的生长，因此我们可以确定哪些蛋白质导致了这种转换。在EP液中，有一种蛋白质比其他所有蛋白质都突出，我们将首先研究这种蛋白质，因为它是最有可能转化的候选蛋白。最令人兴奋的方法是通过x射线衍射来确定其三维结构，这是格拉斯哥的蛋白质组擅长的技术。通过了解它的结构，我们可以确定它是如何工作的。那么，这一切有什么好处呢？好吧，两个重要的方面：首先，如果你可以控制这种开关，你可以依次在许多不同的基板上铺设不同的层（我们也将研究）。有证据表明，文石珠层可以潜在地用于刺激骨质疏松症的骨生成，例如，如果可以以合适的形式提供合成珠层。其次，文石珠层的物理特性（极高的硬度）也可以在许多方面得到利用/保护脆弱的表面和人类。

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.