An Oligomeric C‑RING Nacre Protein Influences
Prenucleation Events and Organizes Mineral Nanoparticles
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Abstract
The
mollusk shell nacre layer integrates mineral phases with macromolecular
components such as intracrystalline proteins. However, the roles performed
by intracrystalline proteins in calcium carbonate nucleation and subsequent
postnucleation events (e.g., organization of mineral deposits) in
the nacre layer are not known. We find that AP7, a nacre intracrystalline
C-RING protein, self-assembles to form amorphous protein oligomers
and films on mica that further assemble into larger aggregates or
phases in the presence of Ca<sup>2+</sup>. Using solution nuclear
magnetic resonance spectroscopy, we determine that the protein assemblies
are stabilized by interdomain interactions involving the aggregation-prone
T31–N66 C-terminal C-RING domain but are destabilized by the
labile nature of the intrinsically disordered D1–T19 AA N-terminal
sequence. Thus, the dynamic, amorphous nature of the AP7 assemblies
can be traced to the molecular behavior of the N-terminal sequence.
Using potentiometric methods, we observe that AP7 protein phases prolong
the time interval for prenucleation cluster formation but neither
stabilize nor destabilize ACC clusters. Time-resolved flow cell scanning
transmission electron microscopy mineralization studies confirm that
AP7 protein phases delay the onset of nucleation and assemble and
organize mineral nanoparticles into ring-shaped branching clusters
in solution. These phenomena are not observed in protein-deficient
assays. We conclude that C-RING AP7 protein phases modulate the time
period for early events in nucleation and form strategic associations
with forming mineral nanoparticles that lead to mineral organization