Using sequence data to predict the self-assembly of supramolecular collagen structures.

Collagen fibrils are the major constituents of the extracellular matrix, which provides structural support to vertebrate connective tissues. It is widely assumed that the superstructure of collagen fibrils is encoded in the primary sequences of the molecular building blocks. However, the interplay between large-scale architecture and small-scale molecular interactions makes the ab initio prediction of collagen structure challenging. Here, we propose a model that allows us to predict the periodic structure of collagen fibers and the axial offset between the molecules, purely on the basis of simple predictive rules for the interaction between amino acid residues. With our model, we identify the sequence-dependent collagen fiber geometries with the lowest free energy and validate the predicted geometries against the available experimental data. We propose a procedure for searching for optimal staggering distances. Finally, we build a classification algorithm and use it to scan 11 data sets of vertebrate fibrillar collagens, and predict the periodicity of the resulting assemblies. We analyzed the experimentally observed variance of the optimal stagger distances across species, and find that these distances, and the resulting fibrillar phenotypes, are evolutionary well preserved. Moreover, we observed that the energy minimum at the optimal stagger distance is broad in all cases, suggesting a further evolutionary adaptation designed to improve the assembly kinetics. Our periodicity predictions are not only in good agreement with the experimental data on collagen molecular staggering for all collagen types analyzed, but also for synthetic peptides. We argue that, with our model, it becomes possible to design tailor-made, periodic collagen structures, thereby enabling the design of novel biomimetic materials based on collagen-mimetic trimers.

A 43 Ca nuclear magnetic resonance perspective on octacalcium phosphate and its hybrid derivatives.

43 Ca nuclear magnetic resonance (NMR) spectroscopy has been extensively applied to the detailed study of octacalcium phosphate (OCP), Ca8 (HPO4 )2 (PO4 )4 .5H2 O, and hybrid derivatives involving intercalated metabolic acids (viz., citrate, succinate, formate, and adipate). Such phases are of importance in the development of a better understanding of bone structure. High-resolution 43 Ca magic angle spinning (MAS) experiments, including double-rotation (DOR) 43 Ca NMR, as well as 43 Ca{1 H} rotational echo DOR (REDOR) and 31 P{43 Ca} REAPDOR NMR spectra, were recorded on a 43 Ca-labeled OCP phase at very high magnetic field (20 T), and complemented by ab initio calculations of NMR parameters using the Gauge-Including Projector Augmented Wave-density functional theory (GIPAW-DFT) method. This enabled a partial assignment of the eight inequivalent Ca2+ sites of OCP. Natural-abundance 43 Ca MAS NMR spectra were then recorded for the hybrid organic-inorganic derivatives, revealing changes in the 43 Ca lineshape. In the case of the citrate derivative, these could be interpreted on the basis of computational models of the structure. Overall, this study highlights the advantages of combining high-resolution 43 Ca NMR experiments and computational modeling for studying complex hybrid biomaterials.

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.

Collagen Structure-Function Relationships from Solid-State NMR Spectroscopy.

The extracellular matrix of a tissue is as important to life as the cells within it. Its detailed molecular structure defines the environment of a tissue's cells and thus their properties, including differentiation and metabolic status. Collagen proteins are the major component of extracellular matrices. Self-assembled collagen fibrils provide both specific mechanical properties to handle external stresses on tissues and, at the molecular level, well-defined protein binding sites to interact with cells. How the cell-matrix interactions are maintained against the stresses on the tissue is an important and as yet unanswered question. Similarly, how collagen molecular and fibrillar structures change in aging and disease is a crucial open question. Solid-state NMR spectroscopy offers insight into collagen molecular conformation in intact in vivo and in vitro tissues, and in this Account we review how NMR spectroscopy is beginning to provide answers to these questions. In vivo 13C,15N labeling of the extracellular matrix has given insight into collagen molecular dynamics and generated multidimensional NMR "fingerprints" of collagen molecular structure that allow comparison of local collagen conformation between tissues. NMR studies have shown that charged collagen residues (Lys, Arg) adopt extended-side-chain conformations in the fibrillar structure to facilitate charge-charge interactions between neighboring collagen molecules, while hydrophobic residues (Leu, Ile) fold along the collagen molecular axis to minimize the hydrophobic area exposed to surrounding water. Detailed NMR and molecular modeling work has shown that the abundant Gly-Pro-Hyp (Hyp = hydroxyproline) triplets in collagen triple helices confer well-defined flexibility because the proline is conformationally metastable, in contrast to the expectation that these triplets confer structural rigidity. The alignment of the Gly-Pro-Hyp triplets within the fibril structure means that the Gly-Pro-Hyp molecular flexibility generates fibril flexibility. The fibrillar bands of Gly-Pro-Hyp are highly correlated with collagen ligand binding sites, leading to the hypothesis that the fibril alignment of Gly-Pro-Hyp triplets is essential to protect collagen-ligand binding against external stresses on the tissue. Non-enzymatic chemistry between collagen side-chain amine groups (Lys, Arg) and reducing sugars-glycation-is an important source of matrix structural change in aging and disease. Glycation leads to stiffening of collagen fibrils, which is widely speculated to be the result of intermolecular cross-linking. The chemistry of non-enzymatic glycation has been extensively detailed through NMR studies and has been shown to lead to side-chain modifications as the majority reaction products, rather than intermolecular cross-links, with resultant molecular misalignment in the fibrils. Thus, a picture is beginning to emerge in which collagen glycation causes stiffening through misalignment of collagen molecular flexible regions rather than intermolecular cross-linking, meaning that new thinking is needed on how to alleviate collagen structural changes in aging and disease.

Solid state NMR - An indispensable tool in organic-inorganic biocomposite characterization; refining the structure of octacalcium phosphate composites with the linear metabolic di-acids succinate and adipate.

Octacalcium phosphate (OCP; Ca8(HPO4)2(PO4)4. 5H2O) is a plausible precursor phase of biological hydroxyapatite, which composites with a number of biologically relevant organic metabolites. Widely used material science physicochemical structure determination techniques successfully characterize the mineral component of these composites but leave details of the structure, and interactions with mineral, of the organic component almost completely obscure. The metabolic linear di-acids succinate (SUC) and adipate (ADI) differentially expand the hydrated (100) layer of OCP. 13C13C correlation (proton driven spin diffusion, PDSD) experiments on OCP composited with (U-13C4)-SUC, and (U13C6)-ADI, show that the two di-acids per unit cell adopt non-centrosymmetric but mutually identical structures. 13C{31P}, rotational echo double resonance (REDOR) shows that one end of each linear di-acid is displaced further from the surface of the apatitic OCP layer relative to the other end. Overall the results indicate two di-acids per unit cell disposed perpendicularly across the OCP hydrated layer with one carboxylate of each di-acid substituting a hydrated surface OCP phosphate group. This study re-affirms the unique advantages of ssNMR in elucidating structural details of organic-inorganic biocomposites, and thereby mechanisms underlying the roles of small metabolites in influencing biomineralization mechanisms and outcomes.

Tuning hardness in calcite by incorporation of amino acids.

Structural biominerals are inorganic/organic composites that exhibit remarkable mechanical properties. However, the structure-property relationships of even the simplest building unit-mineral single crystals containing embedded macromolecules-remain poorly understood. Here, by means of a model biomineral made from calcite single crystals containing glycine (0-7 mol%) or aspartic acid (0-4 mol%), we elucidate the origin of the superior hardness of biogenic calcite. We analysed lattice distortions in these model crystals by using X-ray diffraction and molecular dynamics simulations, and by means of solid-state nuclear magnetic resonance show that the amino acids are incorporated as individual molecules. We also demonstrate that nanoindentation hardness increased with amino acid content, reaching values equivalent to their biogenic counterparts. A dislocation pinning model reveals that the enhanced hardness is determined by the force required to cut covalent bonds in the molecules.

Citrate bridges between mineral platelets in bone.

We provide evidence that citrate anions bridge between mineral platelets in bone and hypothesize that their presence acts to maintain separate platelets with disordered regions between them rather than gradual transformations into larger, more ordered blocks of mineral. To assess this hypothesis, we take as a model for a citrate bridging between layers of calcium phosphate mineral a double salt octacalcium phosphate citrate (OCP-citrate). We use a combination of multinuclear solid-state NMR spectroscopy, powder X-ray diffraction, and first principles electronic structure calculations to propose a quantitative structure for this material, in which citrate anions reside in a hydrated layer, bridging between apatitic layers. To assess the relevance of such a structure in native bone mineral, we present for the first time, to our knowledge, (17)O NMR data on bone and compare them with (17)O NMR data for OCP-citrate and other calcium phosphate minerals relevant to bone. The proposed structural model that we deduce from this work for bone mineral is a layered structure with thin apatitic platelets sandwiched between OCP-citrate-like hydrated layers. Such a structure can explain a number of known structural features of bone mineral: the thin, plate-like morphology of mature bone mineral crystals, the presence of significant quantities of strongly bound water molecules, and the relatively high concentration of hydrogen phosphate as well as the maintenance of a disordered region between mineral platelets.

Solid state NMR of isotope labelled murine fur: a powerful tool to study atomic level keratin structure and treatment effects.

We have prepared mouse fur extensively 13C,15N-labelled in all amino acid types enabling application of 2D solid state NMR techniques which establish covalent and spatial proximities within, and in favorable cases between, residues. 13C double quantum-single quantum correlation and proton driven spin diffusion techniques are particularly useful for resolving certain amino acid types. Unlike 1D experiments on isotopically normal material, the 2D methods allow the chemical shifts of entire spin systems of numerous residue types to be determined, particularly those with one or more distinctively shifted atoms such as Gly, Ser, Thr, Tyr, Phe, Val, Leu, Ile and Pro. Also the partial resolution of the amide signals into two signal envelopes comprising of α-helical, and ß-sheet/random coil components, enables resolution of otherwise overlapped α-carbon signals into two distinct cross peak families corresponding to these respective secondary structural regions. The increase in resolution conferred by extensive labelling offers new opportunities to study the chemical fate and structural environments of specific atom and amino acid types under the influence of commercial processes, and therapeutic or cosmetic treatments.

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.

Citrate occurs widely in healthy and pathological apatitic biomineral: mineralized articular cartilage, and intimal atherosclerotic plaque and apatitic kidney stones.

There is continuing debate about whether abundant citrate plays an active role in biomineralization of bone. Using solid state NMR dipolar dephasing, we examined another normally mineralized hard tissue, mineralized articular cartilage, as well as biocalcifications arising in pathological conditions, mineralized intimal atherosclerotic vascular plaque, and apatitic uroliths (urinary stones). Residual nondephasing ¹³C NMR signal at 76 ppm in the spectra of mineralized cartilage and vascular plaque indicates that a quaternary carbon atom resonates at this frequency, consistent with the presence of citrate. The presence, and as yet unproven possible mechanistic involvement, of citrate in tissue mineralization extends the compositional, structural, biogenetic, and cytological similarities between these tissues and bone itself. Out of 10 apatitic kidney stones, five contained NMR-detectable citrate. Finding citrate in a high proportion of uroliths may be significant in view of the use of citrate in urolithiasis therapy and prophylaxis. Citrate may be essential for normal biomineralization (e.g., of cartilage), play a modulatory role in vascular calcification which could be a target for therapeutic intervention, and drive the formation of apatitic rather than other calcific uroliths, including more therapeutically intractable forms of calcium phosphate.

Lipids in biocalcification: contrasts and similarities between intimal and medial vascular calcification and bone by NMR.

Pathomechanisms underlying vascular calcification biogenesis are still incompletely understood. Biomineral from human atherosclerotic intimal plaques; human, equine, and bovine medial vascular calcifications; and human and equine bone was released from collagenous organic matrix by sodium hydroxide/sodium hypochlorite digestion. Solid-state (13)C NMR of intimal plaque mineral shows signals from cholesterol/cholesteryl esters and fatty acids. In contrast, in mineral from pure medial calcifications and bone mineral, fatty acid signals predominate. Refluxing (chloroform/methanol) intimal plaque calcifications removes the cholesterylic but not the fatty acyl signals. The lipid composition of this refluxed mineral now closely resembles that of the medial and bone mineral, which is unchanged by reflux. Thus, intimal and medial vascular calcifications and bone mineral have in common a pool of occluded mineral-entrained fatty acyl-rich lipids. This population of fatty acid may contain methyl-branched fatty acids, possibly representing lipoprotein particle remnants. Cell signaling and mechanistic parallels between physiological (orthotopic) and pathological (ectopic) calcification are also reflected thus in the NMR spectroscopic fingerprints of mineral-associated and mineral-entrained lipids. Additionally the atherosclerotic plaque mineral alone shows a significant independent pool of cholesterylic lipids. Colocalization of mineral and lipid may be coincidental, but it could also reflect an essential mechanistic component of biomineralization.

Applications of NMR crystallography to problems in biomineralization: refinement of the crystal structure and 31P solid-state NMR spectral assignment of octacalcium phosphate.

By combining X-ray crystallography, first-principles density functional theory calculations, and solid-state nuclear magnetic resonance spectroscopy, we have refined the crystal structure of octacalcium phosphate (OCP), reassigned its (31)P NMR spectrum, and identified an extended hydrogen-bonding network that we propose is critical to the structural stability of OCP. Analogous water networks may be related to the critical role of the hydration state in determining the mechanical properties of bone, as OCP has long been proposed as a precursor phase in bone mineral formation. The approach that we have taken in this paper is broadly applicable to the characterization of crystalline materials in general, but particularly to those incorporating hydrogen that cannot be fully characterized using diffraction techniques.

Apatite in kidney stones is a molecular composite with glycosaminoglycans and proteins: evidence from nuclear magnetic resonance spectroscopy, and relevance to Randall's plaque, pathogenesis and prophylaxis.

PURPOSE: We characterized the biomacromolecular composition of phosphatic urinary stones using solid state nuclear magnetic resonance spectroscopy. We identified possible parallels between the nature of the organic matrix-mineral interface in stones and that in other mineralized tissue using nuclear magnetic resonance spectroscopy rotational echo double resonance. MATERIALS AND METHODS: We analyzed 28 phosphatic (apatite and mixed apatite-struvite) surgically removed stones by nuclear magnetic resonance spectroscopy using (31)P, (13)C and a 9.4 Tesla magnetic field. Ten samples had sufficient signal from biomacromolecular organic material to characterize the mineral/organic interface by (13)C{(31)P} rotational echo double resonance. RESULTS: Biomacromolecular organic material was most abundant in phosphatic stones in which apatite predominated. Nuclear magnetic resonance spectroscopy detected variable proportions of protein, glycosaminoglycan, lipid and carbonate. Rotational echo double resonance revealed strong interaction between mineral and glycosaminoglycan molecules, and to a lesser extent protein molecules, on the sub-nm length scale, implying that glycosaminoglycan and protein are composited into or onto the mineral lattice by strong physicochemical interactions. Carbonate ions substituted into apatite crystal lattices also showed the expected strong (13)C{(31)P} rotational echo double resonance effects. Conversely when present, lipid, calcium oxalate hydrates and uric acid showed no rotational echo double resonance effects, proving that they exist as deposits or crystals distinct from phosphatic mineral/biomacromolecular composites. CONCLUSIONS: The intimate coexistence of biomacromolecules, especially glycosaminoglycan, with apatite in phosphatic stones supports the notion that they may have a key role in stone pathogenesis. The underlying intermolecular relationships may reflect those governing the formation of Randall's plaque in nascent stones.

Collagen atomic scale molecular disorder in ochronotic cartilage from an alkaptonuria patient, observed by solid state NMR.

UNLABELLED: In pilot studies of the usefulness of solid state nuclear magnetic resonance spectroscopy in characterizing chemical and molecular structural effects of alkaptonuria on connective tissue, we have obtained (13) C spectra from articular cartilage from an AKU patient. An apparently normal anatomical location yielded a cross polarization magic angle spinning spectrum resembling literature spectra and dominated by collagen and glycosaminoglycan signals. All spectral linewidths from strongly pigmented ochronotic cartilage however were considerably increased relative to the control indicating a marked increase in collagen molecular disorder. This disordering of cartilage structural protein parallels, at the atomic level, the disordering revealed at higher length scales by microscopy. We also demonstrate that the abnormal spectra from ochronotic cartilage fit with the abnormality in the structure of collagen fibres at the ultrastructural level, whereby large ochronotic deposits appear to alter the structure of the collagen fibre by invasion and cross linking. SUMMARY: Increased signal linewidths in solid state NMR spectra of ochronotic articular cartilage from an AKU patient relative to linewidths in normal, control, cartilage reveals a marked decrease in collagen molecular order in the diseased tissue. This atomic level disordering parallels higher length scale disorder revealed by microscopic techniques.

Tannin fingerprinting in vegetable tanned leather by solid state NMR spectroscopy and comparison with leathers tanned by other processes.

Solid state ¹³C-NMR spectra of pure tannin powders from four different sources--mimosa, quebracho, chestnut and tara--are readily distinguishable from each other, both in pure commercial powder form, and in leather which they have been used to tan. Groups of signals indicative of the source, and type (condensed vs. hydrolyzable) of tannin used in the manufacture are well resolved in the spectra of the finished leathers. These fingerprints are compared with those arising from leathers tanned with other common tanning agents. Paramagnetic chromium (III) tanning causes widespread but selective disappearance of signals from the spectrum of leather collagen, including resonances from acidic aspartyl and glutamyl residues, likely bound to Cr (III) structures. Aluminium (III) and glutaraldehyde tanning both cause considerable leather collagen signal sharpening suggesting some increase in molecular structural ordering. The ²7Al-NMR signal from the former material is consistent with an octahedral coordination by oxygen ligands. Solid state NMR thus provides easily recognisable reagent specific spectral fingerprints of the products of vegetable and some other common tanning processes. Because spectra are related to molecular properties, NMR is potentially a powerful tool in leather process enhancement and quality or provenance assurance.

Incorporation of nanogels within calcite single crystals for the storage, protection and controlled release of active compounds.

Nanocarriers have tremendous potential for the encapsulation, storage and delivery of active compounds. However, current formulations often employ open structures that achieve efficient loading of active agents, but that suffer undesired leakage and instability of the payloads over time. Here, a straightforward strategy that overcomes these issues is presented, in which protein nanogels are encapsulated within single crystals of calcite (CaCO3). Demonstrating our approach with bovine serum albumin (BSA) nanogels loaded with (bio)active compounds, including doxorubicin (a chemotherapeutic drug) and lysozyme (an antibacterial enzyme), we show that these nanogels can be occluded within calcite host crystals at levels of up to 45 vol%. Encapsulated within the dense mineral, the active compounds are stable against harsh conditions such as high temperature and pH, and controlled release can be triggered by a simple reduction of the pH. Comparisons with analogous systems - amorphous calcium carbonate, mesoporous vaterite (CaCO3) polycrystals, and calcite crystals containing polymer vesicles - demonstrate the superior encapsulation performance of the nanogel/calcite system. This opens the door to encapsulating a broad range of existing nanocarrier systems within single crystal hosts for the efficient storage, transport and controlled release of various active guest species.

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.

Probing the calcium and sodium local environment in bones and teeth using multinuclear solid state NMR and X-ray absorption spectroscopy.

Despite the numerous studies of bone mineral, there are still many questions regarding the exact structure and composition of the mineral phase, and how the mineral crystals become organised with respect to each other and the collagen matrix. Bone mineral is commonly formulated as hydroxyapatite, albeit with numerous substitutions, and has previously been studied by (31)P and (1)H NMR, which has given considerable insight into the complexity of the mineral structure. However, to date, there has been no report of an NMR investigation of the other major component of bone mineral, calcium, nor of common minority cations like sodium. Here, direct analysis of the local environment of calcium in two biological apatites, equine bone (HB) and bovine tooth (CT), was carried out using both (43)Ca solid state NMR and Ca K-edge X-ray absorption spectroscopy, revealing important structural information about the calcium coordination shell. The (43)Ca delta(iso) in HB and CT is found to correlate with the average Ca-O bond distance measured by Ca K-edge EXAFS, and the (43)Ca NMR linewidths show that there is a greater distribution in chemical bonding around calcium in HB and CT, compared to synthetic apatites. In the case of sodium, (23)Na MAS NMR, high resolution 3Q-MAS NMR, as well as (23)Na{(31)P} REDOR and (1)H{(23)Na} R(3)-HMQC correlation experiments give the first direct evidence that some sodium is located inside the apatite phase in HB and CT, but with a greater distribution of environments compared to a synthetic sodium substituted apatite (Na-HA).

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

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.

The mineral phase of calcified cartilage: its molecular structure and interface with the organic matrix.

We have studied the atomic level structure of mineralized articular cartilage with heteronuclear solid-state NMR, our aims being to identify the inorganic species present at the surfaces of the mineral crystals which may interact with the surrounding organic matrix and to determine which components of the organic matrix are most closely involved with the mineral crystals. One-dimensional (1)H and (31)P and two-dimensional (1)H-(31)P heteronuclear correlation NMR experiments show that the mineral component is very similar to that in bone with regard to its surface structure. (13)C{(31)P} rotational echo double resonance experiments identify the organic molecules at the mineral surface as glycosaminoglycans, which concurs with our recent finding in bone. There is also evidence of gamma-carboxyglutamic acid residues interacting with the mineral. However, other matrix components appear more distant from the mineral compared with bone. This may be due to a larger hydration layer on the mineral crystal surfaces in calcified cartilage.