Designing Interfaces Between Inorganic Crystals and De Novo Proteins

Hybrid materials composed of biomacromolecules and inorganic crystals have exceptional properties and fulfill critical functional requirements of the organisms that produce them. These biominerals are known to contain proteins that play roles in controlling the crystal polymorph, shape, size, and organization of the mineral constituents. However, the structural details of protein-inorganic interfaces inside biominerals remain largely unknown and are challenging to study because native biomineralization proteins are often intrinsically disordered and function within complex multi-component systems. Designing well-structured de novo proteins with surfaces that form favorable interactions with inorganic crystals has allowed us to study both how these interfaces promote adsorption and assembly of proteins on existing minerals, and how the proteins template mineral growth.<br/>Previously, we have shown that designed helical repeat proteins (DHR) with a periodic array of side chains lattice matched to the mica surface absorbed and assembled into 2D structures at the mica-water interface. The structure of the interfacial assemblies could be modulated by adding protein-protein interfaces in addition to the protein-mica interface. The size, regularity, and stable rod-like shape of these proteins has enabled further study on the effects of solvent and electrolytes on biomolecules at the solid liquid interface and mapping of the angular energy landscape of adsorbed proteins.<br/>Recently, we have shown that specific DHRs that contain designed periodic arrays of negatively charged carboxylate containing side chains promote the direct nucleation of the calcite polymorph of calcium carbonate (CaCO3). This bypasses the formation of vaterite which is seen in the absence of protein, in the presence of bovine serum albumin control protein, and in the presence of a DHR with a similar surface but a different distance between adjacent repeats. Mutational analysis of a protein that promotes calcite formation indicates that activity depends on sidechain stereochemistry and is not driven by charge alone. The calcite nucleating proteins also altered the post-nucleation growth mechanism from classical growth to oriented particle attachment, a mechanism often seen in natural biomineralization processes.<br/>Currently, we are working with a new designed library of over 6.5K de novo repeat proteins displayed on the surface of yeast. These proteins have a variety of topologies and have diverse chemical groups arranged in periodic and aperiodic arrays on their surfaces. We are using a high throughput flow-cytometry (FC) assay to screen for proteins that promote the strong adsorption of inorganic nanoparticles to the yeast cells displaying them. Nanoparticle binding proteins have been selected from the library and expressed in E coli, purified, and biochemically characterized, and we are studying their effects on the growth of these materials from ionic precursors and on metallic substrates.<br/>Overall, our research shows the potential of de novo protein design as a powerful tool for studying and manipulating the interfaces between genetically encoded proteins and inorganic crystals, offering exciting prospects for biomimetic materials synthesis.