The formation of crack propagation—resistant single crystal calcite in the prismatic layer of the mollusk shell involves the participation of a number of different proteins, some of which form intracrystalline organic inclusions. One protein family, Asprich (Atrina rigida), participate in the ACC formation/transformation process and become occluded within calcite. However, these two phenomena are poorly understood. Here, we experimentally establish that the Asprich “3” protein oligomerizes in solution over a wide pH range and that this oligomerization process is enhanced by the presence of Ca2+ ions, which form complexes with this protein as verified by ESI-MS. Bioinformatics analyses confirm that intrinsic disorder and unstable interactive domains constitute the entire length of each Asprich sequence. In addition, an amyloid- or prion-like aggregation-prone region was identified within the highly conserved 12 AA N-terminal sequence, KPVFKRSLSDPS. Together, these bioinformatics findings suggest that different sequence elements of the Asprich family contribute to the observed oligomerization process. Solution NMR studies of Asprich “3” protein assemblies document that portions of the 12 AA N-terminal domain are mobile within these assemblies. This mobility is driven by Pro imido ring cis-trans isomerization at P11, and this induces multiple conformational states for V3, R6, and L8 which lie upstream of this Pro residue. This Asprich “3” Pro-induced conformational exchange phenomena parallels other findings obtained for self-assembling nacre layer- and tooth enamel-specific biomineralization proteins. In conclusion, we have identified the presence of self-association, unusual sequence behavior, and ion clustering phenomena in Asprich “3” and we believe that these are key factors in the ability of Asprich proteins to control ACC formation and form organic inclusions that resist crack propagation.
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