NMR Spectroscopy of Native and in Vitro Tissues Implicates PolyADP Ribose in Biomineralization

Abstract

Nuclear magnetic resonance (NMR) spectroscopy is useful to determine molecular structure in tissues grown in vitro only if their fidelity, relative to native tissue, can be established. Here, we use multidimensional NMR spectra of animal and in vitro model tissues as fingerprints of their respective molecular structures, allowing us to compare the intact tissues at atomic length scales. To obtain spectra from animal tissues, we developed a heavy mouse enriched by about 20% in the NMR-active isotopes carbon-13 and nitrogen-15. The resulting spectra allowed us to refine an in vitro model of developing bone and to probe its detailed structure. The identification of an unexpected molecule, poly(adenosine diphosphate ribose), that may be implicated in calcification of the bone matrix, illustrates the analytical power of this approach.

Structure of fully protonated proteins by proton-detected magic-angle spinning NMR

“…their effective use could be applied to a much wider array of molecules including biomolecules labeled in mammalian systems (44). However, although amide proton assignment is possible for fully protonated proteins above 40-kHz MAS (45)(46)(47)(48), proton resonances remain significantly dipolar broadened at 40-60 kHz, limiting the applicability of this spinning regime for side-chain assignment and structure determination (41,45,(49)(50)(51).…”

The contribution of solid-state NMR spectroscopy to understanding biomineralization: Atomic and molecular structure of bone

“…Thus, our first work with our NMR-based molecular fingerprints of native bone tissue was to develop an in vitro tissue that at the atomic scale, as judged by NMR and at the nanoscopic lengthscale as judged by SEM and AFM (Fig. 4), was as similar as possible to native tissue [45]. Interestingly, the differences between NMR signals we found from in vitro grown tissues and those from native tissues were from proline-/ glycine-rich peptides with 13 C chemical shifts consistently higher than those for a collagen triple helix structure.…”

“…Bone collagen had one of the highest levels of enrichment using this approach, between 80% and 90%. Our aim here [45] was to be able to record multidimensional correlation NMR spectra of intact, native tissues in which the full chemical shift distributions of all essential amino acid 13 C and 15 N were resolved and correlated with the chemical shift distributions of all spatially-close 13 C and 15 N. We argued that a set of such spectra, correlating both 13 C with 13 C and 13 C with 15 N, and for different spectral mixing times, i.e. different upper internuclear distance constraints on the observed spectral correlations, together constituted a ''fingerprint'' of the underlying molecular structures.…”

Preparation of highly and generally enriched mammalian tissues for solid state NMR

“…tryptophan, in the algal hydrolysate (see Supplementary Table S1 for comparison of amino acid contents of the two feedstocks). In the rapid turnover bone collagen, feeding with labelled diet during adulthood only produces 2.9-fold spectral enhancement, which corresponds to sufficient labelling for structurally meaningful 2D spectroscopy (Chow et al 2014). The meagre isotope enrichment of only 1.6-fold produced by adult feeding in fur keratin (typifying a slow turnover tissue) is quite insufficient for this purpose.…”

“…In previously published work (Chow et al 2014) we achieved levels of 13 C labelling in some tissues, sufficient for high quality 2D NMR, by simply feeding labelled diet to an adult mouse for about three weeks. Using this protocol, however, the level of labelling varied widely between different tissue types, being among the highest in bone collagen, and barely detectable in slower turnover biomaterials such as fur keratin, among others, precluding any structurally informative 2D experiments in the latter.…”