Structural description of surfaces and interfaces in biominerals by DNP SENS

Abstract

Biological mineralized tissues are hybrid materials with complex hierarchical architecture composed of biominerals often embedded in an organic matrix. The atomic-scale comprehension of surfaces and organo-mineral interfaces of these biominerals is of paramount importance to understand the ultrastructure, the formation mechanisms as well as the biological functions of the related biomineralized tissue. In this communication we demonstrate the capability of DNP SENS to reveal the fine atomic structure of biominerals, and more specifically their surfaces and interfaces. For this purpose, we studied two key examples belonging to the most significant biominerals family in nature: apatite in bone and aragonite in nacreous shell. As a result, we demonstrate that DNP SENS is a powerful approach for the study of intact biomineralized tissues. Signal enhancement factors are found to be up to 40 and 100, for the organic and the inorganic fractions, respectively, as soon as impregnation time with the radical solution is long enough (between 12 and 24 h) to allow an efficient radical penetration into the calcified tissues. Moreover, ions located at the biomineral surface are readily detected and identified through 31 P or 13 C HETCOR DNP SENS experiments. Noticeably, we show that protonated anions are preponderant at the biomineral surfaces that are in the form of HPO4 2for bone apatite and HCO3 2for nacreous aragonite. Finally, we demonstrate that organo-mineral interactions can be probed at the atomic level with high sensitivity. In particular, reliable { 31 P}-13 C REDOR experiments are achieved in a few hours, leading to the determination of distances, molar proportion and binding mode of citrate bonded to bone mineral in native compact bone. According to our results, only 50% of the total amount of citrate in bone is directly interacting with bone apatite through two out of three carboxylic groups.

Recent directions in the solid-state NMR study of synthetic and natural calcium phosphates

“…[62] More importantly, it is worth noting that a broadening as well as shifts in some of the NMR resonances were observed on the DNP spectra (in comparison to conventional NMR analyses) ; this was ascribed to the lower temperatures used in the DNP analyses (but without verifying this point by performing a conventional 1 H  13 C CPMAS NMR analysis at 100 K). [81,82] Moreover, differences in DNP enhancements were also noticed for the mineral and organic components of bone (with stronger enhancements for the organic fraction, as shown in Figures 6b-c) ; [81] this implies that future optimizations of the impregnating conditions may also depend on the type of structural information which is sought.…”

“…[66,89] On the other hand, using DNP, it was shown that a full set of 13 C{ 31 P} REDOR dephasing curves could be recorded on a sheep bone sample in just 11 hours (instead of several weeks in absence of DNP), and this allowed estimating the distance of citrate carbon atoms from the mineral surface. [81] In the latter case, based on these dephasing curves, it was suggested that only two out of three carboxylate functions attach to the mineral surface. This level of information is particularly important in relation to the different computational modeling studies of the organic/mineral interface.…”

“…In this case, the need to impregnate the sample by a radical solution raises the question of the choice of the radical/solvent couple, the goal being to avoid (or minimize) any structural changes within the biological sample. When looking at the few studies reported so far, most bone sample preparations involved an AMUPol biradical solution in H2O/D2O, [81][82][83] leading to 13 C DNPenhancement factors between ~ 20 and 60 ( Figure 6). It is worth noting that these impregnation procedures differed slightly in terms of AMUPol concentration (between 12 and 20 mM), D2O/H2O ratio, duration of the impregnation, and size of the bone particles studied.…”

Targetable Tetrazine‐Based Dynamic Nuclear Polarization Agents for Biological Systems

“…Dynamic nuclear polarization (DNP) has had a significant impact on solid‐state nuclear magnetic resonance investigations of complex biological systems . With now routine signal enhancements in the 100‐fold range, DNP has aided the in vitro structural studies of amyloid polymers, membrane proteins, and biological material such as collagen, bones and tissues . Improvements in DNP instrumentation, sample preparation, and polarization agent design have also allowed NMR spectroscopists to turn their attention to the cellular environment, where the structural analysis of endogenous concentrations of biological macromolecules has come within reach .…”

Targetable Tetrazine-Based Dynamic Nuclear Polarization Agents for Biological Systems

“…Dynamic nuclear polarization (DNP) has made significant impact on the solid-state nuclear magnetic resonance (NMR) investigations of complex biological systems. [1][2] With now routine signal enhancements in the 100-fold range, DNP has aided the in vitro structural studies of amyloid polymers, [3][4] membrane proteins [5][6] and biological material such as collagen, bones and tissues [7][8] . Improvements in DNP instrumentation, sample preparation and polarization agent design have also allowed NMR spectroscopists to turn their attention to the cellular environment where the structural analysis of endogenous concentrations of biological macromolecules has come within reach.…”