Ca 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, Ca-complexes are dynamic soluble entities challenging to describe at the molecular level. Nuclear magnetic resonance appears as a method of choice to probe Ca-complexes. However, Ca NMR exhibits severe limitations arising from the low natural abundance coupled to the low gyromagnetic ratio and the quadrupolar nature of Ca, which overall make it a very unreceptive nucleus. Here, we show that Ca dynamic nuclear polarization (DNP) NMR of Ca-labeled frozen solutions is an efficient approach to enhance the NMR receptivity of Ca 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 Ca nuclei belonging to Ca complexed to the investigated ligands exhibit rather low quadrupolar couplings (with 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 H→Ca cross-polarization (CP) is used to observe Ca central transition, "high-power" ν(Ca) 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) H-Ca HetCor spectra to be readily recorded, highlighting various Ca-ligand interactions in solution. This significant increase in Ca NMR sensitivity results from the combination of distinct advantages: (i) an efficient H-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 Ca environments of calcium-containing frozen solution, capable of filtering out more rigid and/or anisotropic Ca 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.