Solid state NMR of salivary calculi: Proline-rich salivary proteins, citrate, polysaccharides, lipids, and organic–mineral interactions

This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.crci.2015.07.001Solid state NMR (ssNMR) can characterize mineral (31P) and organic (13C) components of human salivary stones (n = 8). All show apatitic 31P spectra. 13C ssNMR indicates more protein, of more consistent composition, than apatitic uroliths, with signals from Tyr, Phe and His prominent. Citrate and lipid, identified by dipolar dephasing (DD), and polysaccharides are also observable in varying amounts. 13C{31P} rotational echo double resonance (13C{31P} REDOR) identifies carbon atoms in close (< ca. 0.5 nm) proximity to phosphorus and therefore probably binding with mineral. Citrate, sugar and carboxylate signals undergo strong 13C{31P} REDOR, also seen to signals between 50 and 60 ppm, from protein α- carbons and, possibly, phosphoserines and phospholipids, and sometimes to a 35 – 40 ppm envelope containing Asp-Cβ and Glu-Cγ signals. Amino acid analysis indicates high proline and aromatic content. 13C ssNMR and amino acid composition is consistent with preponderance of proline-rich salivary proteins such as statherin.The U.K. EPSRC (Y. L.) and MRC (D. G. R.) for fundin

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

An appreciable level of isotope labelling is essential for future NMR structure elucidation of mammalian biomaterials, which are either poorly expressed, or unexpressable, using micro-organisms. We present a detailed protocol for high level (13)C enrichment even in slow turnover murine biomaterials (fur keratin), using a customized diet supplemented with commercial labelled algal hydrolysate and formulated as a gel to minimize wastage, which female mice consumed during pregnancy and lactation. This procedure produced approximately eightfold higher fur keratin labelling in pups, exposed in utero and throughout life to label, than in adults exposed for the same period, showing both the effectiveness, and necessity, of this approach.The authors would like to acknowledge funding from the Biotechnology and Biological Sciences Research Council for DGR and RR; Engineering and Physical Sciences Research Council for WYC and VWCW; National Institute of Health Research for RAB.This is the final version of the article. It first appeared from Springer via http://dx.doi.org/10.1007/s10858-015-9977-

Hydroxyproline Ring Pucker Causes Frustration of Helix Parameters in the Collagen Triple Helix.

Collagens, the most abundant proteins in mammals, are defined by their triple-helical structures and distinctive Gly-Xaa-Yaa repeating sequence, where Xaa is often proline and Yaa, hydroxyproline (Hyp/O). It is known that hydroxyproline in the Yaa position stabilises the triple helix, and that lack of proline hydroxylation in vivo leads to dysfunctional collagen extracellular matrix assembly, due to a range of factors such as a change in hydration properties. In addition, we note that in model peptides, when Yaa is unmodified proline, the Xaa proline has a strong propensity to adopt an endo ring conformation, whilst when Yaa is hydroxyproline, the Xaa proline adopts a range of endo and exo conformations. Here we use a combination of solid-state NMR spectroscopy and potential energy landscape modelling of synthetic triple-helical collagen peptides to understand this effect. We show that hydroxylation of the Yaa proline causes the Xaa proline ring conformation to become metastable, which in turn confers flexibility on the triple helix.The authors acknowledge BBSRC grant number BB/G021392/1 (MJD, DGR), EPSRC DTA studentship and Doctoral Prize (WYC), British Heart Foundation RG/09/003/27122 and PG/08/011/24416 (RWF, DB, DAS), Wellcome Trust 094470/Z/10/Z (RWF, DB, DAS), ERC and EPSRC (DJW).This is the final version of the article. It first appeared from NPG via http://dx.doi.org/10.1038/srep1255

The curious case of (caffeine).(benzoic acid): how heteronuclear seeding allowed the formation of an elusive cocrystal

Cocrystals are modular multicomponent solids with exceptional utility in synthetic chemistry and materials science. A variety of methods exist for the preparation of cocrystals yet, some promising cocrystal phases have proven to be intractable synthetic targets. We describe a strategy for the synthesis of the pharmaceutically relevant (caffeine).(benzoic acid) cocrystal (1), which persistently failed to form using a broad range of established techniques. State-of-the-art crystal structure prediction methods were employed to assess the possible existence of a thermodynamically stable form of 1, and to identify appropriate heteronuclear seeds for corystallization. Once introduced, the designed heteronuclear seeds facilitated the formation of 1 and, significantly, continued to act as long-lasting laboratory .contaminants., which encouraged cocrystal formation even when present at such low levels as to evade detection. The seeding technique described thus enables the synthesis of cocrystals regarded as unobtainable under desired conditions, and potentially signifies a new direction in the field of materials research

Collagen labelling with an azide-proline chemical reporter in live cells.

We have developed a strategy for selective imaging of collagen in live foetal ovine osteoblasts. Our approach involves the incorporation of an azide-tagged proline in the biosynthesis of collagen followed by labelling using a strain-promoted [3+2] azide-alkyne cycloaddition reaction

Proline provides site-specific flexibility for in vivo collagen.

Fibrillar collagens have mechanical and biological roles, providing tissues with both tensile strength and cell binding sites which allow molecular interactions with cell-surface receptors such as integrins. A key question is: how do collagens allow tissue flexibility whilst maintaining well-defined ligand binding sites? Here we show that proline residues in collagen glycine-proline-hydroxyproline (Gly-Pro-Hyp) triplets provide local conformational flexibility, which in turn confers well-defined, low energy molecular compression-extension and bending, by employing two-dimensional 13C-13C correlation NMR spectroscopy on 13C-labelled intact ex vivo bone and in vitro osteoblast extracellular matrix. We also find that the positions of Gly-Pro-Hyp triplets are highly conserved between animal species, and are spatially clustered in the currently-accepted model of molecular ordering in collagen type I fibrils. We propose that the Gly-Pro-Hyp triplets in fibrillar collagens provide fibril "expansion joints" to maintain molecular ordering within the fibril, thereby preserving the structural integrity of ligand binding sites.BBSRC, EPSRC, Raymond and Beverly Sackler Fund for Physics of Medicine, Wellcome Trust, ER

NMR spectroscopy of native and in vitro tissues implicates polyADP ribose in biomineralization.

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

Mechanical adaptation of brachiopod shells via hydration-induced structural changes.

The function-optimized properties of biominerals arise from the hierarchical organization of primary building blocks. Alteration of properties in response to environmental stresses generally involves time-intensive processes of resorption and reprecipitation of mineral in the underlying organic scaffold. Here, we report that the load-bearing shells of the brachiopod Discinisca tenuis are an exception to this process. These shells can dynamically modulate their mechanical properties in response to a change in environment, switching from hard and stiff when dry to malleable when hydrated within minutes. Using ptychographic X-ray tomography, electron microscopy and spectroscopy, we describe their hierarchical structure and composition as a function of hydration to understand the structural motifs that generate this adaptability. Key is a complementary set of structural modifications, starting with the swelling of an organic matrix on the micron level via nanocrystal reorganization and ending in an intercalation process on the molecular level in response to hydration

Pigmentation Chemistry and Radical-Based Collagen Degradation in Alkaptonuria and Osteoarthritic Cartilage

Alkaptonuria (AKU) is a rare disease characterized by high levels of homogentisic acid (HGA); patients suffer from tissue ochronosis: dark brown pigmentation, especially of joint cartilage, leading to severe early osteoarthropathy. No molecular mechanism links elevated HGA to ochronosis; the pigment's chemical identity is still not known, nor how it induces joint cartilage degradation. Here we give key insight on HGA-derived pigment composition and collagen disruption in AKU cartilage. Synthetic pigment and pigmented human cartilage tissue both showed hydroquinone-resembling NMR signals. EPR spectroscopy showed that the synthetic pigment contains radicals. Moreover, we observed intrastrand disruption of collagen triple helix in pigmented AKU human cartilage, and in cartilage from patients with osteoarthritis. We propose that collagen degradation can occur via transient glycyl radicals, the formation of which is enhanced in AKU due to the redox environment generated by pigmentation

Glycation changes molecular organization and charge distribution in type I collagen fibrils

Funder: Royal Society Newton TrustFunder: China Scholarship Council Cambridge TrustsFunder: SENS Research Foundation; doi: http://dx.doi.org/10.13039/100013772Funder: Raymond and Beverly Sackler Fund for Physics of MedicineFunder: Engineering and Physical Sciences Research Council; doi: http://dx.doi.org/10.13039/501100000266Abstract: Collagen fibrils are central to the molecular organization of the extracellular matrix (ECM) and to defining the cellular microenvironment. Glycation of collagen fibrils is known to impact on cell adhesion and migration in the context of cancer and in model studies, glycation of collagen molecules has been shown to affect the binding of other ECM components to collagen. Here we use TEM to show that ribose-5-phosphate (R5P) glycation of collagen fibrils – potentially important in the microenvironment of actively dividing cells, such as cancer cells – disrupts the longitudinal ordering of the molecules in collagen fibrils and, using KFM and FLiM, that R5P-glycated collagen fibrils have a more negative surface charge than unglycated fibrils. Altered molecular arrangement can be expected to impact on the accessibility of cell adhesion sites and altered fibril surface charge on the integrity of the extracellular matrix structure surrounding glycated collagen fibrils. Both effects are highly relevant for cell adhesion and migration within the tumour microenvironment