Characterization of a Ca(II)-, mineral-interactive polyelectrolyte sequence from the adhesive elastomeric biomineralization protein lustrin A

The lustrin protein superfamily represents a class of elastomeric biomineralization proteins which are localized between layered aragonite mineral plates (i.e., nacre layers) in mollusk shell. These proteins exhibit adhesive behavior within the mineralized matrix, suggesting that there may be interfacial interactions between lustrin proteins and aragonite mineral surfaces and/or other surfaces of organic matrix components. Previously, within one lustrin protein (Lustrin A) we identified a 24 amino acid Asp-rich polyelectrolyte sequence, named D4, which adopts an open, unfolded conformation in solution. This sequence contains a putative Ca(II) interaction sequence, -DTDADSGSD-, which potentially represents a site for Ca(II)mediated Lustrin A-mineral or Lustrin A-organic matrix interactions. In this present study, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, circular dichroism, and nuclear magnetic resonance spectroscopy, we confirm that the D4 sequence is a Ca(II)-binding polypeptide that maintains its unfolded conformation in the presence of Ca(II) ions. Moreover, using geological calcite overgrowth assays and scanning electron microscopy visualization, we observe interruption in calcium carbonate crystal growth in the presence of nanomolar levels of D4 polypeptide. Proton solid-state nuclear magnetic resonance experiments verify that during the course of calcium carbonate crystal growth D4 becomes bound to calcite fragments and cannot be entirely displaced from the mineral fragments using low and high ionic strength conditions. We conclude from our studies that D4 represents a potential matrix adhesion site within the Lustrin A protein and possesses molecular traits that coincide with other reported calcium carbonate mineral interaction domains.