PerspectiveStructural Biology

Choosing the Crystallization Path Less Traveled

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Science  12 Aug 2005:
Vol. 309, Issue 5737, pp. 1027-1028
DOI: 10.1126/science.1114920

A good way to grow large crystals in desired shapes is by slow cooling of a melt phase of the substance on a small crystal seed. The alternative is to perform the same process with a crystal seed and a slightly supersaturated solution. The crystal seed operates as a nucleating substrate that will reliably induce a crystal of choice to form. Without a nucleating substrate, it is very difficult to control formation of the product. When spontaneous crystallization starts, the process can follow several different pathways that inevitably result in a mass of small crystals of various sizes and shapes. The crystals formed by organisms are usually not at all like that; they are of uniform size and shape and are often aligned. So it would seem that biological systems have opted for the nucleation substrate strategy. And so they do. But biological processes cannot operate at the high temperatures required for a melt phase in inorganic minerals. The supersaturated solution strategy could be viable, but because the solubility of mineral crystals is inevitably low, large solution volumes must be removed to form even relatively small crystals. Amazingly, living organisms have found solutions to both of these problems.

The hallmark of the biological strategy for making certain mineralized skeletal parts is producing the first-formed solid deposits as disordered and often hydrated phases that with time transform into the stable crystalline deposit. The first proven example of such a process in a living organism is from the chiton, a mollusk that has mineralized teeth that are used for scraping rocks to extract algae buried beneath the surface. The outer layer of the tooth contains magnetite, a hard magnetic mineral (1). It forms from a disordered ferrihydrite precursor phase (2). The inner layer of the tooth contains carbonated apatite, the same mineral present in bone. It forms by way of an amorphous calcium phosphate precursor phase (3). In the years following these discoveries, it was shown that ferrihydrite is a precursor phase of magnetite formation in magnetotactic bacteria (4). Beniash et al. (5) later reported that the larva of the sea urchin, an echinoderm, forms its calcitic spicule from an amorphous calcium carbonate precursor phase. Mollusk larvae have also been found to form their aragonitic shells from such a precursor phase (6, 7). And adult sea urchins also apparently follow the same protocol for generating their carbonate skeleton (8). Because mollusks and echinoderms are on two different branches of the animal phylogenetic tree, it seems likely that an amorphous calcium carbonate strategy for forming crystals may well be widely used. Indeed, it has been suggested that corals and crustaceans use this approach to produce their skeletons as well (9, 10).

Why should organisms use such a strategy? One reason is that a disordered phase can easily be molded into any shape, whereas a crystal has a strong propensity to adopt a specific shape dictated by the structure of its atomic lattice. There are many examples of beautifully sculpted carbonate minerals in biology (see the figure). If these are all formed by way of an amorphous calcium carbonate precursor phase, the strategy is most certainly widespread. Smooth curved surfaces in biogenic minerals are often the telltale signs of the involvement of cellular membranes in determining shape.

Skeletons from crystals.

Different organisms likely use the same strategy to generate diverse skeletal parts from crystals that arise from a transient amorphous calcium carbonate phase.

CREDIT: S. WEINER ET AL.

For about a century it has been known that organisms can produce amorphous calcium carbonate in a stable form. However, amorphous calcium carbonate is inherently unstable. It was only recently synthesized in vitro as a stable phase, and even then under extreme pH conditions (11). Specific proteins can induce the formation of stable amorphous calcium carbonate in vitro under nearly physiological pH and temperature conditions (12). But stable amorphous calcium carbonate phases differ from each other in that their local atomic organization around the calcium ions varies. Nevertheless, they all have one mole of water per mole of calcium carbonate.

The transient forms of amorphous calcium carbonate that have been observed in organisms are even more enigmatic. They have little or no water, and the nascent short-range order around their calcium ions resembles the calcitic or aragonitic stable phase into which the amorphous phase will transform. All known biogenic amorphous calcium carbonates contain magnesium and phosphate ions (11), suggesting critical importance, yet their roles are not clear. In vitro studies of amorphous calcium carbonate formation point to the stabilizing properties of small confined spaces delimited by a membrane or in a hydrophobic medium (13). Size itself can be important when nano-sized particles are involved (14), as is the case with the spherical subunits that make up amorphous calcium carbonate. Isolating nano-sized amorphous calcium carbonate particles from the external medium until crystallization is desired may well be part of the biological strategy. It may stabilize the transient amorphous phase by preventing contact with water and by preventing contact of the nanoparticles with nucleating substrates.

Crystallization by way of a transient precursor amorphous phase does not preclude the involvement of a designed nucleation substrate. The two processes are clearly integrated in the formation of the sea urchin larval spicule (see the figure). The first step is the oriented nucleation of a single crystal of calcite at a specific location within a large vesicle delimited by a membrane (called a syncitium). The vesicle is then loaded with amorphous calcium carbonate, and a single crystal continues to grow at the expense of the amorphous phase until the entire spicule becomes a single crystal of calcite (11). It is conceivable that fusion of membrane-coated amorphous calcium carbonate particles with the large vesicle membrane delivers them uncoated into the vesicle cavity, thus initiating their transformation into crystals. In mollusk shells, the function of a hydrophobic silk fibroin gel phase may be to prevent crystallization until the particles contact the nucleating substrate, ensuring that oriented crystals will form only at the correct location.

We thus come to the peculiar conclusion that organisms achieve their aim of building large single crystals of desired shape by slowly growing them on a small crystal seed or a nucleation substrate by way of a disordered phase. This disordered phase has characteristics similar to a melt phase but does not require high temperatures. This knowledge may well prove useful for fabricating complex-shaped crystalline synthetic materials; because no water is present in the precursor phase, these materials may also have low porosity and improved mechanical properties.

Is it possible that vertebrates also use the transient precursor phase strategy for forming bones and teeth? This option was discussed in the 1970s after it was discovered that in vitro, under nearly physiological conditions, carbonated apatite (the mineral in bone) forms from an amorphous calcium phosphate precursor phase. After it was shown that this phase is not present in mature bone, the possibility of carbonated apatite formation by way of transient precursor phases was also rejected [reviewed in (15)]. It may be time to reconsider.

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