* These authors contributed equally # Correspondence and request for materials should be directed to JJDY (jjdeyoreo@lbl.gov).Nucleation in the natural world often occurs in the presence of organic interfaces. In mineralized tissues, a range of macromolecular matrices are found in contact with inorganic phases and are believed to direct mineral formation. In geochemical settings, mineral surfaces, which are often covered with organic or biological films, surround the volume within which nucleation occurs.In the classical picture of nucleation, the presence of such interfaces is expected to have a profound effect on nucleation rates, simply because they can reduce the interfacial free energy, which controls the height of the thermodynamic barrier to nucleation of the solid phase.However, the recent discovery of a nearly monodisperse population of calcium carbonate clusters Ñ so called pre-nucleation clusters Ñ and the many observations of amorphous precursor phases have called into question the applicability of classical descriptions. Here we use in situ observations of nucleation on organothiol self-assembled monolayers (SAMs) to explore the energetics and pathways of calcite nucleation at organic interfaces. We find that carboxyl SAM-directed nucleation is described well in purely classical terms through a reduction in the thermodynamic barrier due to decreased interfacial free energy. Moreover, the differences 2 in nucleation kinetics on odd and even chain-length carboxyl SAMs are attributable to relative differences in these energies. These differences arise from varying degrees of SAM order related to oxygen-oxygen interactions between SAM headgroups. In addition, amorphous particles formed prior to or during crystal nucleation do not grow and are not observed to act as precursors to the crystalline phase. Instead, calcite nucleates independently. These results imply that the recently proposed model of calcite formation as a non-classical process, one which proceeds via aggregation of stable pre-nucleation clusters that form an amorphous precursor from which the crystalline phase emerges, is not applicable to template-directed nucleation and does not provide a universal description of calcite formation.
Calcified skeletons are produced within complex assemblages of proteins and polysaccharides whose roles in mineralization are not well understood. Here we quantify the kinetics of calcite nucleation onto a suite of high-purity polysaccharide (PS) substrates under controlled conditions. The energy barriers to nucleation are PS-specific by a systematic relationship to PS charge density and substrate structure that is rooted in minimization of the competing substrate-crystal and substrate-liquid interfacial energies. Chitosan presents a low-energy barrier to nucleation because its near-neutral charge favors formation of a substrate-crystal interface, thus reducing substrate interactions with water. Progressively higher barriers are measured for negatively charged alginates and heparin that favor contact with the solution over the formation of new substrate-crystal interfaces. The findings support a directing role for PS in biomineral formation and demonstrate that substrate-crystal interactions are one end-member in a larger continuum of competing forces that regulate heterogeneous crystal nucleation.biomineralization | calcium carbonate | free energy | algae | crustacean E fforts to decipher patterns of biomineralization have identified proteins and polysaccharides (PSs) as major components of the organic matrices associated with sites of calcification. In mollusks and other organisms including the red algae, coccolithophores, and foraminifera (1, 2) (Table 1), calcifying macromolecules are dominated by functional groups with an acidic character-proteins that are rich in carboxylated amino acids (14) and PSs that are highly sulfated and carboxylated (15,16). Although a physical picture of these interactions is not well developed, this recurring affiliation of carboxylate and sulfate groups with zones of mineralization in organisms suggests specific roles for macromolecules in nucleation and growth. Early studies have led the biomineralization community to generally assume that charged proteins actively regulate mineralization. In contrast, PSs, such as the chitin found in the insoluble fraction of the mollusk shell, are thought to provide an inert scaffolding to support these proteins (17,18).Recent studies challenge this assumption with qualitative evidence that PSs can also promote calcium carbonate (CaCO 3 ) mineralization (19,20) (Table 1). For example, specific orientations of chitin fibers with neutral functional groups promote the templating of CaCO 3 in the lobster carapace (13, 21), crab cuticle (21), and nautilus shell (22). More generally, the monosaccharide sequences along PS chains can influence biological function and their interactions with proteins (23). This suggests PS chemistry and interactions with proteins could regulate patterns of mineralization.Anecdotal observations from in vitro studies also support the thinking that PSs influence mineral formation, but their specific effects are unclear. For example, PSs with higher carboxyl and sulfate content promote either faster (24, 25) or slower (2...
The physical basis for how macromolecules regulate the onset of mineral formation in calcifying tissues is not well established. A popular conceptual model assumes the organic matrix provides a stereochemical match during cooperative organization of solute ions. In contrast, another uses simple binding assays to identify good promoters of nucleation. Here, we reconcile these two views and provide a mechanistic explanation for template-directed nucleation by correlating heterogeneous nucleation barriers with crystal-substrate-binding free energies. We first measure the kinetics of calcite nucleation onto model substrates that present different functional group chemistries (carboxyl, thiol, phosphate, and hydroxyl) and conformations (C11 and C16 chain lengths). We find rates are substrate-specific and obey predictions of classical nucleation theory at supersaturations that extend above the solubility of amorphous calcium carbonate. Analysis of the kinetic data shows the thermodynamic barrier to nucleation is reduced by minimizing the interfacial free energy of the system, γ. We then use dynamic force spectroscopy to independently measure calcitesubstrate-binding free energies, ΔG b . Moreover, we show that within the classical theory of nucleation, γ and ΔG b should be linearly related. The results bear out this prediction and demonstrate that low-energy barriers to nucleation correlate with strong crystal-substrate binding. This relationship is general to all functional group chemistries and conformations. These findings provide a physical model that reconciles the long-standing concept of templated nucleation through stereochemical matching with the conventional wisdom that good binders are good nucleators. The alternative perspectives become internally consistent when viewed through the lens of crystal-substrate binding.B iological systems are unique in their ability to organize minerals into functional materials with complex patterns and architectures. A substantial body of evidence suggests specialized macromolecules, particularly proteins (1, 2) and carbohydrates (3, 4), provide preferential sites for nucleation to direct the placement, timing, and orientation of crystals (5), both intra-and extracellular. Within the biomineralization community, the conventional view of biologically directed nucleation is that macromolecular matrices present an interfacial match to the crystal lattice that assists in forming the crystal nucleus. This cooperative view of directed nucleation is rooted in the collective action of multiple residues that guide the organization of ions into a configuration defining the energetic minimum for the system. A series of in vitro observations have reinforced this picture by showing that highly ordered organic monolayers can control the location and orientation of calcite crystals precipitated from solution (6). In this approach, good templates are revealed through a direct functional assay, i.e., nucleation. Over the years, this view of mineralization, both in the context of natural stru...
A series of Mg‐bearing synthetic amorphous calcium carbonates (ACC) were characterized by Raman spectroscopy. The spectra showed a systematic increase in the carbonate ν1 peak position from the control samples that contained 0.0 mol % MgCO3 to samples that contained up to 43 mol % MgCO3. The relationship is best described by the function: mol % MgCO3 = (ν1 – 1079.66) / 0.2017. The Mg content is equally well‐predicted by a correlation with the instrumentally corrected ν1 full width at half maximum that is quantified by: mol % MgCO3 = (ν1 – 23.26) / 0.1969. An analysis of the Raman data collected for ACC combined with insights from crystalline materials suggests that compositional dependencies arise from changes in the local metal–oxygen bonds as Mg substitutes for Ca. The calibrations described here provide a rapid and nondestructive means of determining the Mg content of ACC, with additional advantages of minimal sample preparation and a high degree of lateral spatial resolution (approximately 1 µm). This method may be appropriate for investigations of heterogeneous samples such as biominerals. Copyright © 2011 John Wiley & Sons, Ltd.
The aspartate-rich macromolecules found at nucleation sites of calcifying organisms are widely implicated in regulating biomineral formation. Anecdotal evidence suggests that their ability to influence the onset of nucleation and composition of calcified structures may arise from effects on ion hydration. This study investigates the interactions of acidic amino acids and dipeptides with hydrated cations using molecular dynamics. By monitoring the hydration states of Mg2+, Ca2+, and Sr2+ during their approach to negatively charged molecules, we show that carboxylate moieties of Asp promote dehydration of Ca2+ and Sr2+. A contact ion pair (CIP) is not required to disrupt cation hydration, and we demonstrate that reductions and rearrangements of first shell water can begin at ion-Asp separation distances as large as approximately 4.9 A for Ca2+ and approximately 5.1 A for Sr2+. CIP formation between Ca2+ and Sr2+ and carboxylate groups decreases the total first shell coordination number from an average of 8.0 and 8.4 in bulk water to 7.5 and 8.0, respectively. The energy barrier to physically replacing waters about Ca2+ with carboxylate oxygen atoms is small (approximately 2 kcal/mol) as compared to a somewhat larger barrier for Sr2+ (approximately 4 kcal/mol). This may be explained by differences in the strength of Coulombic interactions between the cations and the Asp, resulting in different paths of approach toward Asp for Ca2+ and Sr2+. In contrast, the primary solvation shell of Mg2+ remains largely unchanged during interactions with Asp until the abrupt physical replacement of water by carboxylate oxygen atoms, which comes at a high energetic cost. These insights support the claim that carboxylated biomolecules increase the growth rate of calcite by lowering the energy barrier to Ca2+ dehydration. The findings also suggest a physical basis for the idea that ion-specific behaviors of Ca2+ and Mg2+ in cellular systems arise from a critical balance between water binding in the ion hydration shells versus their interactions with ligands present in intracellular environments.
The carbonate sedimentary record contains diverse compositions and textures that reflect the evolution of oceans and atmospheres through geological time. Efforts to reconstruct paleoenvironmental conditions from these deposits continue to be hindered by the need for process-based models that can explain observed shifts in carbonate chemistry and form. Traditional interpretations assume minerals precipitate and grow by classical ion-by-ion addition processes but are unable to reconcile a number of unusual features contained in Proterozoic carbonates. The realization that diverse organisms produce high Mg carbonate skeletal structures by non-classical pathways involving amorphous intermediates raises the question of whether similar processes are also active in sedimentary environments. This study examines the hypothesis that non-classical pathways to mineralization are the physical basis for some of the carbonate morphologies and compositions observed in natural and laboratory settings. We designed experiments with a series of different solution Mg : Ca ratios and saturation environments to investigate the effects on carbonate phase, Mg content, and morphology. Our observations of diverse carbonate mineral compositions and textures suggest geochemical conditions bias the mineralization pathway by a systematic relationship to Mg : Ca ratio and the abundance of carbonate ions. Environments with low Mg levels produce calcite crystallites with 0-12 mol% MgCO 3 . In contrast, the combination of high initial Mg : Ca and rapidly increasing saturation opens a non-classical pathway that begins with extensive precipitation of an amorphous calcium carbonate (ACC). This phase slowly transforms to aggregates of very high Mg calcite nanoparticles whose structures and compositions are similar to natural disordered dolomites. The non-classical pathways are favored when the local environment contains sufficient Mg to inhibit calcite growth through increased solubility-a thermodynamic factor, and achieves saturation with respect to ACC on a timescale that is shorter than the rate of aragonite nucleation-a kinetic factor. Aragonite is produced when Mg levels are high but saturation is insufficient for ACC precipitation. The findings provide a physical basis for anecdotal claims that the interplay of kinetic and thermodynamic factors underlies patterns of carbonate precipitation and suggest the need to expand traditional interpretations of geological carbonate formation to include non-classical pathways to mineralization.
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