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)-bi...