Molluskan nacre is a biocomposite consisting to ∼95% of the aragonitic polymorph of calcium carbonate and an organic matrix of polysaccharide and protein (1–4). Nacre has a fracture toughness three orders of magnitude higher than the pure mineral, a tensile strength of 140–170 MPa, and a Young's modulus of 60–70 GPa. These extraordinary physical properties are due to the structure and composition of the biocomposite. The mineral phase of abalone nacre consists of vertical stacks of flat polygonal aragonite tablets which form brick-wall-like horizontal sheets of lamellae. Each tablet is encased by an organic matrix which leaves pores for mineral bridges connecting the tablets of a stack to form a single crystal (5,6). The organic matrix thus guides crystal growth to form the characteristic brick and mortar construction of nacre and acts as an adhesive, filling the space between aragonite crystals as shown by atomic force microscopy (AFM) (7).
A major component of the insoluble fraction of the matrix is the polysaccharide chitin (8,9), which may act as a scaffold in the interlamellar organic sheets. The first protein from abalone nacre characterized by sequence analysis was the large multidomain protein lustrin A (10), which has been shown to be a component of the adhesive between aragonite tablets (7). Recently two other proteins, perlucin and perlustrin, were isolated from nacre and characterized at the molecular level (11). Perlucin is a C-type lectin with galactose/mannose binding specificity (12) which nucleates new layers in a calcium carbonate crystal when added to calcite crystals and precipitates together with calcium carbonate crystals in in vitro assays (13). Perlustrin is an insulin- and insulin-like growth factor-binding protein (14), the presence of which in nacre may be related to earlier findings (15) that extracts of nacre stimulate proliferation and differentiation processes of vertebrate fibroblasts, bone-marrow cells, and osteoblasts.
The AFM (16) is a valuable tool to investigate dynamic processes on mineral surfaces and protein-mineral interactions on the micro- and nanometer scale. The time frame of AFM imaging is compatible with the speed of growth and dissolution of molecular layers of calcite crystals, and therefore this method can be used to study the effect of different solvents on surface layers of the calcium carbonate polymorph calcite (17). It was also possible to observe the interaction of a mixture of water-soluble nacre proteins with calcite, and it was shown that binding of such proteins can change the shape of atomic step edges on the calcite crystal surface and the speed of crystal growth (18). The protein mixture also induced the transition from calcite to aragonite when applied to a growing calcite surface (19). In this report, we describe the isolation and sequence analysis of perlwapin, an abalone nacre protein consisting of three consecutive WAP domains, and the effect of the purified protein on calcium carbonate crystallization as shown by AFM in aqueous solution.