In situ molecular NMR picture of bioavailable calcium stabilized as amorphous CaCO₃ biomineral in crayfish gastroliths

Abstract

Bioavailable calcium is maintained by some crustaceans, in particular freshwater crayfish, by stabilizing amorphous calcium carbonate (ACC) within reservoir organs--gastroliths, readily providing the Ca(2+) needed to build a new exoskeleton. Despite the key scientific and biomedical importance of the in situ molecular-level picture of biogenic ACC and its stabilization in a bioavailable form, its description has eluded efforts to date. Herein, using multinuclear NMR, we accomplish in situ molecular-level characterization of ACC within intact gastroliths of the crayfish Cherax quadricarinatus. In addition to the known CaCO(3), chitin scaffold and inorganic phosphate (Pi), we identify within the gastrolith two primary metabolites, citrate and phosphoenolpyruvate (PEP) and quantify their abundance by applying solution NMR techniques to the gastrolith "soluble matrix." The long-standing question on the physico-chemical state of ACC stabilizing, P-bearing moieties within the gastrolith is answered directly by the application of solid state rotational-echo double-resonance (REDOR) and transferred-echo double-resonance (TEDOR) NMR to the intact gastroliths: Pi and PEP are found molecularly dispersed throughout the ACC as a solid solution. Citrate carboxylates are found < 5 Å from a phosphate (intermolecular CP distance), an interaction that must be mediated by Ca(2+). The high abundance and extensive interactions of these molecules with the ACC matrix identify them as the central constituents stabilizing the bioavailable form of calcium. This study further emphasizes that it is imperative to characterize the intact biogenic CaCO(3). Solid state NMR spectroscopy is shown to be a robust and accessible means of determining composition, internal structure, and molecular functionality in situ.

Fig. 1.

¹³C MAS NMR spectra of intact gastrolith of the crayfish C. quadricarinatus (A and B) and of the decalcified gastrolith (C). (A) DE MAS, fully relaxed spectrum (40-min repetition delay); (B) CP MAS spectrum and its vertical expansion (× 4) with peak assignment of the bioorganic content. (C) CP MAS spectrum of the decalcified gastrolith showing the insoluble matrix. (Top) Molecular structures of the chitin repeat unit and of PEP and citrate. (D and E) Photos of the intact and of the decalcified gastrolith.

Fig. 2.

(A) ³¹P NMR spectra. (Bottom) 121.5-MHz CP MAS spectrum of intact gastrolith of C. quadricarinatus showing two partially resolved peaks assigned to Pi and PEP. (Top) 161.3-MHz solution NMR of the soluble matrix. Solution chemical shift values of phosphates are pH dependent and are different from the in situ values (SI Text). (B) ³¹P → ¹³C 2D-HETCOR MAS NMR spectrum of intact gastrolith of the crayfish C. quadricarinatus showing connectivity between Pi to ACC carbonate and between PEP phosphate to ACC carbonate. ¹H to ³¹P contact time of 2 ms and 24 × 24T_R TEDOR mixing were employed. (C) A schematic drawing of ACC with Pi and PEP molecularly dispersed.

Fig. 3.

¹³C{³¹P} CP-REDOR MAS NMR spectra (64T_R; 12.8 ms) of intact gastrolith of the crayfish C. quadricarinatus. (A) S₀ reference spectrum: accounts for all carbon species; (B) ΔS difference spectrum: shows peaks only for species that are within 9 Å from a P atom of Pi or PEP. (C) Expanded carbonate region: shows a 50 ± 2% REDOR difference peak. (D) Experimental (points; two gastroliths) and simulated REDOR ΔS/S₀ evolution curves; simulations are for 1∶1 CaCO₃∶H₂O spheres (R = 7, 8, 9, 10 Å) with a central P atom, accounting for ¹³C⋯³¹P distance distribution (SI Text).