Protein-mineral interactions and inorganic nucleation through designed protein interfaces

Abstract

Biomolecules have the ability to regulate the formation of hierarchically structured biominerals through their interactions with inorganic crystals. However, the details of the atomic structure at the organic-inorganic interface that governs this process are not yet known. This work will present a set of design principles for the creation of molecular templates targeted to interact with calcium carbonate and hematite. The starting hypothesis states that a structured flat molecular template could achieve heterogeneous nucleation of calcium carbonate or facet-specific and oriented binding of proteins to hematite by pre-organizing binding moieties for calcium or iron on its surface. To test this, helical repeat proteins displaying regularly spaced carboxylate arrays on their surfaces were designed. It was discovered that these protein templates directly nucleate nano calcite with non-natural (110) or (202) facets. These proteins also allow for the bypassing of vaterite, which forms in the absence of the proteins. The resulting nanocrystals then come together by oriented attachment to form calcite mesocrystals. By altering the protein length and manipulating their surface chemistry, the nanocrystal size and nucleation rate can be adjusted. As the size of the carboxylate arrays decreased, the nanocrystal diameters increased. Furthermore, the nucleation activity was eliminated by partially replacing the carboxylates with lysines. In the case of hematite, binding studies suggested that proteins with a target spacing of 10.9 Å (but a most likely interhelical spacing of 11.2 Å) are capable of interacting specifically with the target (012) surface. This was observed for incubation conditions under which the surface potential is identical for both test facets. Binding on the off-target (001) surface was heterogeneous and domain dependent. Nucleation studies of hematite in the presence of proteins suggested that several designed and non-designed proteins had a degree of inhibition in the effect of nucleation of hematite starting with a ferrihydrite precursor. In sum, these templates achieve a degree of tunability only accessible through protein design and represent one of the most programmable systems for broader biomineralization studies. These advances open the possibility to use de novo protein design to program biomineralization, offering a pathway to creating advanced hybrid materials.