Samuel Lenton

Mineralisation of soft and hard tissues and the stability of biofluids

Evidence is provided from studies on natural and artificial biofluids that the sequestration of amorphous calcium phosphate by peptides or proteins to form nanocluster complexes is of general importance in the control of physiological calcification. A naturally occurring mixture of osteopontin peptides was shown, by light and neutron scattering, to form calcium phosphate nanoclusters with a core-shell structure. In blood serum and stimulated saliva, an invariant calcium phosphate ion activity product was found which corresponds closely in form and magnitude to the ion activity product observed in solutions of these osteopontin nanoclusters. This suggests that types of nanocluster complexes are present in these biofluids as well as in milk. Precipitation of amorphous calcium phosphate from artificial blood serum, urine and saliva was determined as a function of pH and the concentration of osteopontin or casein phosphopeptides. The position of the boundary between stability and precipitation was found to agree quantitatively with the theory of nanocluster formation. Artificial biofluids were prepared that closely matched their natural counterparts in calcium and phosphate concentrations, pH, saturation, ionic strength and osmolality. Such fluids, stabilised by a low concentration of sequestering phosphopeptides, were found to be highly stable and may have a number of beneficial applications in medicine.

A review of the biology of calcium phosphate sequestration with special reference to milk.

In milk, a stable fluid is formed in which sequestered nanoclusters of calcium phosphate are substructures in casein micelles. As a result, calcium and phosphate concentrations in milk can be far in excess of their solubility. Variations of calcium, phosphate and casein concentrations in milks, both within and among species, are mainly due to the formation of the nanocluster complexes. Caseins evolved from tooth and bone proteins well before the evolution of lactation. It has therefore been suggested that the role of caseins in milk is an adaptation of an antecedent function in the control of some aspect of biomineralisation. There is new evidence that nanocluster-type complexes are also present in blood serum and, by implication, in many other closely related biofluids. Because such fluids are stable but nevertheless supersaturated with respect to the bone and tooth mineral hydroxyapatite, they allow soft and mineralised tissues to co-exist in the same organism with relative ease. An appreciable concentration of nanocluster complexes exists in fresh saliva. Such saliva may stabilise tooth mineral and help to repair demineralised lesions. In the extracellular matrix of bone, nanocluster complexes may be involved in directing the amorphous calcium phosphate to intrafibrillar spaces in collagen where they can mature into oriented apatite crystals. Thus, evidence is accumulating that calcium phosphate sequestration by phosphopeptides to form equilibrium complexes, first observed in milk, is more generally important in the control of physiological calcification.

Dynamic footprint of sequestration in the molecular fluctuations of osteopontin

The sequestration of calcium phosphate by unfolded proteins is fundamental to the stabilization of biofluids supersaturated with respect to hydroxyapatite, such as milk, blood or urine. The unfolded state of osteopontin (OPN) is thought to be a prerequisite for this activity, which leads to the formation of core–shell calcium phosphate nanoclusters. We report on the structures and dynamics of a native OPN peptide from bovine milk, studied by neutron spectroscopy and small-angle X-ray and neutron scattering. The effects of sequestration are quantified on the nanosecond– ångström resolution by elastic incoherent neutron scattering. The molecular fluctuations of the free phosphopeptide are in agreement with a highly flexible protein. An increased resilience to diffusive motions of OPN is corroborated by molecular fluctuations similar to those observed for globular proteins, yet retaining conformational flexibilities. The results bring insight into the modulation of the activity of OPN and phosphopeptides with a role in the control of biomineralization. The quantification of such effects provides an important handle for the future design of new peptides based on the dynamics–activity relationship.

Effect of Phosphorylation on a Human-like Osteopontin Peptide.

The last decade established that the dynamic properties of the phosphoproteome are central to function and its modulation. The temporal dimension of phosphorylation effects remains nonetheless poorly understood, particularly for intrinsically disordered proteins. Osteopontin, selected for this study due to its key role in biomineralization, is expressed in many species and tissues to play a range of distinct roles. A notable property of highly phosphorylated isoforms of osteopontin is their ability to sequester nanoclusters of calcium phosphate to form a core-shell structure, in a fluid that is supersaturated but stable. In Biology, this process enables soft and hard tissues to coexist in the same organism with relative ease. Here, we extend our understanding of the effect of phosphorylation on a disordered protein, the recombinant human-like osteopontin rOPN. The solution structures of the phosphorylated and unphosphorylated rOPN were investigated by small-angle x-ray scattering and no significant changes were detected on the radius of gyration or maximum interatomic distance. The picosecond-to-nanosecond dynamics of the hydrated powders of the two rOPN forms were further compared by elastic and quasi-elastic incoherent neutron scattering. Phosphorylation was found to block some nanosecond side-chain motions while increasing the flexibility of other side chains on the faster timescale. Phosphorylation can thus selectively change the dynamic behavior of even a highly disordered protein such as osteopontin. Through such an effect on rOPN, phosphorylation can direct allosteric mechanisms, interactions with substrates, cofactors and, in this case, amorphous or crystalline biominerals.

Highly compressed water structure observed in a perchlorate aqueous solution

The discovery by the Phoenix Lander of calcium and magnesium perchlorates in Martian soil samples has fueled much speculation that flows of perchlorate brines might be the cause of the observed channeling and weathering in the surface. Here, we study the structure of a mimetic of Martian water, magnesium perchlorate aqueous solution at its eutectic composition, using neutron diffraction in combination with hydrogen isotope labeling and empirical potential structure refinement. We find that the tetrahedral structure of water is heavily perturbed, the effect being equivalent to pressurizing pure water to pressures of order 2 GPa or more. The Mg2+ and ClO4− ions appear charge-ordered, confining the water on length scales of order 9 Å, preventing ice formation at low temperature. This may explain the low evaporation rates and high deliquescence of these salt solutions, which are essential for stability within the low relative humidity environment of the Martian atmosphere.Significant amounts of different perchlorate salts have been discovered on the surface of Mars. Here, the authors show that magnesium perchlorate has a major impact on water structure in solution, providing insight into how an aqueous fluid might exist under the sub-freezing conditions present on Mars.

Assessing the Potential of Folded Globular Polyproteins As Hydrogel Building Blocks

The native states of proteins generally have stable well-defined folded structures endowing these biomolecules with specific functionality and molecular recognition abilities. Here we explore the potential of using folded globular polyproteins as building blocks for hydrogels. Photochemically cross-linked hydrogels were produced from polyproteins containing either five domains of I27 ((I27)5), protein L ((pL)5), or a 1:1 blend of these proteins. SAXS analysis showed that (I27)5 exists as a single rod-like structure, while (pL)5 shows signatures of self-aggregation in solution. SANS measurements showed that both polyprotein hydrogels have a similar nanoscopic structure, with protein L hydrogels being formed from smaller and more compact clusters. The polyprotein hydrogels showed small energy dissipation in a load/unload cycle, which significantly increased when the hydrogels were formed in the unfolded state. This study demonstrates the use of folded proteins as building blocks in hydrogels, and highlights the potential versatility that can be offered in tuning the mechanical, structural, and functional properties of polyproteins.

Temperature-Dependent Segregation in Alcohol–Water Binary Mixtures Is Driven by Water Clustering

Previous neutron scattering work, combined with computer simulated structure analysis, has established that binary mixtures of methanol and water partially segregate into water-rich and alcohol-rich components. It has furthermore been noted that, between methanol mole fractions of 0.27 and 0.54, both components, water and methanol, simultaneously form percolating clusters. This partial segregation is enhanced with decreasing temperature. The mole fraction of 0.27 also corresponds to the point of maximum excess entropy for ethanol-water mixtures. Here, we study the degree of molecular segregation in aqueous ethanol solutions at a mole fraction of 0.27 and compare it with that in methanol-water solutions at the same concentration. Structural information is extracted for these solutions using neutron diffraction coupled with empirical potential structure refinement. We show that ethanol, like methanol, bi-percolates at this concentration and that, in a similar manner to methanol, alcohol segregation, as measured by the proximity of neighboring methyl sidechains, is increased upon cooling the solution. Water clustering is found to be significantly enhanced in both alcohol solutions compared to the water clustering that occurs for random, hard sphere-like, mixing with no hydrogen bonds between molecules. Alcohol clustering via the hydrophobic groups is, on the other hand, only slightly sensitive to the water hydrogen bond network. These results support the idea that it is the water clustering that drives the partial segregation of the two components, and hence the observed excess entropy of mixing.