⁴³Ca MAS-DNP NMR of Frozen Solutions for the Investigation of Calcium Ion Complexation

Calcium ion complexation in aqueous solutions is of paramount importance in biology as it is related to cell signaling, muscle contraction, or biomineralization. However, Ca²⁺-complexes are dynamic soluble entities challenging to describe at the molecular level. Nuclear magnetic resonance appears as a method of choice to probe Ca²⁺-complexes. However, ⁴³Ca NMR exhibits severe limitations arising from the low natural abundance coupled to the low gyromagnetic ratio and the quadrupolar nature of ⁴³Ca, which overall make it a very unreceptive nucleus. Here, we show that ⁴³Ca dynamic nuclear polarization (DNP) NMR of ⁴³Ca-labeled frozen solutions is an efficient approach to enhance the NMR receptivity of ⁴³Ca and to obtain structural insights about calcium ions complexed with representative ligands including water molecules, ethylenediaminetetraacetic acid (EDTA), and l-aspartic acid (l-Asp). In these conditions and in combination with numerical simulations and calculations, we show that ⁴³Ca nuclei belonging to Ca²⁺ complexed to the investigated ligands exhibit rather low quadrupolar couplings (with C typically ranging from 0.6 to 1 MHz) due to high symmetrical environments and potential residual dynamics in vitrified solutions at a temperature of 100 K. As a consequence, when ¹H→⁴³Ca cross-polarization (CP) is used to observe ⁴³Ca central transition, "high-power" ν_RF(⁴³Ca) conditions, typically used to detect spin 1/2 nuclei, provide ∼120 times larger sensitivity than "low-power" conditions usually employed for detection of quadrupolar nuclei. These "high-power" CPMAS conditions allow two-dimensional (2D) ¹H-⁴³Ca HetCor spectra to be readily recorded, highlighting various Ca²⁺-ligand interactions in solution. This significant increase in ⁴³Ca NMR sensitivity results from the combination of distinct advantages: (i) an efficient ¹H-mediated polarization transfer from DNP, resembling the case of low-natural-abundance spin 1/2 nuclei, (ii) a reduced dynamics, allowing the use of CP as a sensitivity enhancement technique, and (iii) the presence of a relatively highly symmetrical Ca environment, which, combined to residual dynamics, leads to the averaging of the quadrupolar interaction and hence to efficient high-power CP conditions. Interestingly, these results indicate that the use of high-power CP conditions is an effective way of selecting symmetrical and/or dynamic ⁴³Ca environments of calcium-containing frozen solution, capable of filtering out more rigid and/or anisotropic ⁴³Ca sites characterized by larger quadrupolar constants. This approach could open the way to the atomic-level investigation of calcium environments in more complex, heterogeneous frozen solutions, such as those encountered at the early stages of calcium phosphate or calcium carbonate biomineralization events.