Abstract
O-phospho-l-serine (Pser) and its Ca salt, Ca[O-phospho-l-serine]·H2O (CaPser), play important roles for bone mineralization and were recently also proposed to account for the markedly improved bone-adhesive properties of Pser-doped calcium phosphate-based cements for biomedical implants. However, the hitherto few proposed structural models of Pser and CaPser were obtained by X-ray diffraction, thereby leaving the proton positions poorly defined. Herein, we refine the Pser and CaPser structures by density functional theory (DFT) calculations and contrast them with direct interatomic-distance constraints from two-dimensional (2D) nuclear magnetic resonance (NMR) correlation experimentation at fast magic-angle spinning (MAS), encompassing double-quantum–single-quantum (2Q–1Q) 1H NMR along with heteronuclear 13C{1H} and 31P{1H} correlation NMR experiments. The Pser and CaPser structures before and after refinements by DFT were validated against sets of NMR-derived effective 1H–1H, 1H–31P, and 1H–13C distances, which confirmed the improved accuracy of the refined structures. Each distance set was derived from one sole 2D NMR experiment applied to a powder without isotopic enrichment. The distances were extracted without invoking numerical spin-dynamics simulations or approximate phenomenological models. We highlight the advantages and limitations of the new distance-extraction procedure. Isotropic 1H, 13C, and 31P chemical shifts obtained by DFT calculations using the gauge including projector augmented wave (GIPAW) method agreed very well with the experimental results. We discuss the isotropic and anisotropic 13C and 31P chemical-shift parameters in relation to the previous literature, where most data on CaPser are reported herein for the first time.
Highlights
We have refined previously reported X-ray diffraction (XRD)-derived crystal structures of Pser and CaPser by density functional theory (DFT) calculations and evaluated both the previous/refined structure options against experimental 1H, 13C, and 31P chemical shifts as well as direct {P−Hk}, {Cj−Hk}, and {Hj−Hk} distance constraints, each extracted by one 2D MAS nuclear magnetic resonanace (NMR) experiment applied to the powdered sample with all isotopes at natural abundance
Whereas relatively modest improvements resulted for the XRD-derived structure of CaPser, much closer agreements with the NMR results were observed for the DFT-refined Pser structure
A decisive advantage of our 2D NMR analysis strategy relative to previously reported NMR crystallography applications for obtaining internuclear distances is the minimum of efforts required both experimentally and numerically: provided that the various 2D correlation NMR signals are reasonably resolved and that a sufficiently short dipolar recoupling period is employed such that eq 14 is obeyed, entire sets of effective interatomic distances are readily obtained from one sole 2D NMR experiment, without any fitting against phenomenological expressions or time-consuming multiple-spin simulations to reproduce the 2D NMR peak-intensity buildup for increasing dipolar recoupling periods, where each such data point demands the recording of one additional 2D NMR spectrum
Summary
Protein residues with negatively charged side chains stemming from either phosphorylation or carboxy groups occur frequently in many non-collagenous proteins (NCPs) believed to govern the growth of bone and tooth mineral.[1−3] Bone mineral consists of a carbonated form of the mineral calcium hydroxyapatite (HA), which associates with fibrils of type I collagen to build the hierarchical bone structure.[2−6] the mechanisms behind bone-mineral formation and how they are initiated and controlled remain heavily debated over decades.[2,3,6] Inarguably, the negatively charged COO− and/orPO42−-bearing residues of NCPs at inorganic calcium phosphate render (CaP)them readily adsorbed surfaces,[1−3,7,8] encompassing bone mineral, HA, and other crystalline as well as structurally disordered CaP phases. One example is the complexes between amorphous calcium phosphate (ACP)[6,9] and casein in milk.[10−13] Strong affinities for binding at CaP surfaces are manifested by small and negatively charged biomolecules, such as amino acids and the ester of L-serine and phosphoric acid, O-phospho-L-serine (Pser); see Figure 1a This feature is confirmed from experimental adsorption studies at (nano)crystalline HA particles[14−19] along with computational modeling,[20−22] encompassing very recent findings on the association of Pser molecules and ACP present in Pserbearing CaP cements (CPCs), which is believed to underpin their bone-adhesive properties.[23,24]. Wu et al.[25] demonstrated that 31P magic-angle-spinning (MAS) nuclear magnetic resonanace (NMR) spectra obtained from 8−12 days old chick embryos revealed signatures of NCP-associated PO4 groups devoid of contacts with Ca2+ in the youngest embryos, whereas those aged for ≥10 days manifested Ca2+···PO42− motifs, similar to those encountered in the Ca salt of phosphoserine, Ca[O-phospho-L-serine]·H2O (CaPser; Figure 1b).[25,26] As highlighted in refs 25 and 27 and discussed further
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