Engineering Proteins and Peptoids to Direct and Accelerate Calcium Carbonate Nucleation and Growth

The extensive deposits of CaCO₃ generated by marine organisms constitute the largest and oldest CO₂ reservoir on the planet. These organisms utilize macromolecules, like proteins, to facilitate the nucleation and growth of carbonate minerals, serving as effective agents of CO₂ sequestration. However, despite the opportunity mineralization presents for extracting anthropogenic CO₂ from the environment, neither the precise mechanisms behind this process nor the design principles required to create potent modulators of carbonate mineralization are known. Here we report on two related research efforts to design proteins and protein-like molecules that direct and accelerate CaCO₃ nucleation and growth: one focused on amphiphilic peptoids that increase calcite growth rates by an order of magnitude, and another directed towards <i>de novo</i> design of proteins to template CaCO₃ nucleation. To understand the mechanism of growth acceleration by peptoids, we used: 1) <i>in situ</i> AFM to measure the growth rates, roughness, fluctuations and critical lengths of atomic steps on calcite as a function of peptoid sequence, supersaturation, and Ca²⁺:CO₃^2- ratio, 2) 3D AFM to probe the effect of peptoids on interfacial solution structure, and 3) liquid state NMR to determine their impact on desolvation and deprotonation rates. The results show that growth acceleration comes primarily from enhanced deprotonation of HCO₃^- combined with an increase in step roughness and step-edge fluctuations, as well as disruption of the interfacial hydration structure. To create proteins that template CaCO₃ nucleation, we designed helical repeat proteins displaying periodic, planar arrays of carboxylates and used <i>in situ</i> TEM and FTIR to investigate their effect on nucleation. The results show that both protein monomers and protein-Ca²⁺ supramolecular assemblies directly nucleate nano-calcite with non-natural {110} or {202} faces while vaterite, which forms first in the absence of the proteins, is bypassed. These protein-stabilized nanocrystals then assemble by oriented attachment into calcite mesocrystals. We find further that nanocrystal size and polymorph can be tuned by varying the length and surface chemistry of the designed protein templates. Taken together, these two studies provide a potential route to design of effective synthetic proteins and biomimetic polymers for removal of CO₂ from the environment via enhanced mineralization.