Polysaccharide controls on the kinetics and thermodynamics of CaCO₃ nucleation: Insights for biological crystallization and other biofunctions

Abstract

Sulfated and carboxylated polysaccharides are ubiquitous in living systems. This class of biomacromolecules modulates diverse biochemical processes from cell signaling to biomineralization, yet our knowledge of how glycomaterials influence biological processes is limited compared to proteins. We particularly lack a quantitative understanding of how differences in the functional group compositions and positions on polysaccharides direct the heterogeneous nucleation of calcite (CaCO₃), an important marine biomineral. At many sites of CaCO₃ biomineralization, anionic polysaccharides are intimately associated with chitin. To establish a broader understanding of macromolecular controls on mineralization, this dissertation is focused on building a quantitative and mechanistic understanding of how polysaccharide materials influence CaCO₃ crystallization. Using advances in biopolymer chemistry, we prepared a series of sulfated chitosan derivatives with varied positions and degrees of sulfation (DS(SO₃⁻)) and measured the rates of CaCO₃ nucleation onto these materials. A dual flow-through system maintained a constant supersaturation for the duration of each experiment which allowed us to determine the interfacial energy barrier (γnet) to calcite nucleation. The measurements established that γnet correlates with sulfate density by a relationship that is independent of sulfate position. We propose that greater sulfate density creates a progressively hydrophilic, negatively charged environment at the polysaccharide-water interface to increase γnet through reductions in γmacromolecule-solution. Such an environment would promote Ca²⁺ interactions with sulfate. To test this idea, we conducted a molecular dynamics (MD) study of sulfated chitosan chains that also have varying degrees and positions of sulfation. The MD simulations show increased water structuring around SO₃⁻ groups compared to uncharged substituents. SO₃⁻-Ca²⁺ interactions are solvent-separated by a distance that decreases with increasing DS(SO₃⁻). The simulations also suggest polysaccharide-Ca²⁺ interactions are dependent on functional group type and position. The interactions of Ca²⁺ with sulfated polysaccharides were further investigated using isothermal titration calorimetry (ITC) and heparin as a model compound for diverse sulfated polysaccharides. Heparin is a glycosaminoglycan of long-standing importance as an anticoagulant in biomedicine. Using heparosan (control) and heparins with different densities and positions of sulfate groups, we show the free energies of the heparin-Ca²⁺ interactions (ΔGrxn) are dominated by entropic contributions (-TΔSrxn) due to favorable water release from polar, hydrophilic groups. We find -TΔSrxn upon Ca²⁺-polysaccharide binding correlates with the γnet for CaCO₃ nucleation, suggesting molecule-specific differences in nucleation rate can be explained by differences in water structuring during Ca²⁺ interactions. The hypothesis that water structuring at polysaccharide surfaces during (solvent-separated) Ca²⁺ interactions drives interfacial energy in CaCO₃ systems suggests CaCO₃ nucleation occurs near (rather than on) a polysaccharide-solution interface at separations that are correlated with hydrophilicity. To test this model, we directly imaged the nucleation of CaCO₃ onto aminated silica nanoparticles (SiO₂-NH₂), chitosan-coated, and heparin-coated SiO₂-NH₂ using in situ liquid-phase transmission electron microscopy (TEM). The method uses silicon nitride (Si₃N₄) membranes to create a closed liquid cell for imaging real-time reactions. We find the polysaccharide-coated nanoparticles create a favorable environment for localized crystallization, promoting CaCO₃ nucleation near the polysaccharide interface rather than in bulk solution. The crystals form in equidistant rings about the polysaccharide-coated nanoparticles, suggesting that nucleation occurs at the polysaccharide- Si₃N₄ membrane-solution interface. We predict that water structuring is most disrupted in this region, resulting in a low γnet, thus favoring crystal nucleation. The combined experimental and computational findings in this dissertation emphasize the importance of relationships between the energy barrier to crystallization, macromolecule composition, and solvent structuring when ions interact with biomacromolecules during biochemical processes. This foundational knowledge furthers our understanding of polysaccharide structure-function controls on mineralization in living systems while also fostering innovations that lead to designing new materials for advanced applications.