DataSheet1_Calcium interactions in amelogenin-derived peptide assembly.pdf

Phosphorylation of serine residues has been recognized as a pivotal event in the evolution of mineralized tissues in many biological systems. During enamel development, the extracellular matrix protein amelogenin is most abundant and appears to be critical to the extreme high aspect ratios (length:width) of apatite mineral fibers reaching several millimeters in larger mammalian teeth. A 14-residue peptide (14P2, residues Gly8 to Thr21) was previously identified as a key sequence mediating amelogenin assembly formation, the domain also contains the native single phosphoserine residue (Ser16) of the full-length amelogenin. In this research, 14P2 and its phosphorylated form (p14P2) were investigated at pH 6.0 with various calcium and phosphate ion concentrations, indicating that both peptides could self-assemble into amyloid-like conformation but with differences in structural details. With calcium, the distance between 31P within the p14P2 self-assemblies is averaged to be 4.4 ± 0.2Å, determined by solid-state NMR 31P PITHIRDS-CT experiments. Combining with other experimental results, solid-state Nuclear Magnetic Resonance (SSNMR) suggests that the p14P2 self-assemblies are in parallel in-register β-sheet conformation and divalent calcium ions most likely connect two adjacent peptide chains by binding to the phosphate group of Ser16 and the carboxylate of Glu18 side-chain. This study on the interactions between calcium ions and amelogenin-derived peptides provides insights on how amelogenin may self-assemble in the presence of calcium ions in early enamel development.</p

Schematic drawing for the sample preparation and visualization under the light microscopy observation and AFM-based nanoindentation analysis.

<p>Schematic drawing for the sample preparation and visualization under the light microscopy observation and AFM-based nanoindentation analysis.</p

FTIR and micro-FTIR analysis of demineralized and remineralized dentin.

<p>A-D) selected individual spectra obtained from area as indicated by arrows, showing peaks associated with phosphate groups at 1020–1160 cm^-1 and around 1660 cm^-1 for amide-I; E) micro-FTIR map of cross section through the demineralized dentin lesion (DE), plotted as area of intensity between 1020–1160 cm^-1; F) micro-FTIR map for range 1020–1160 cm^-1 of DEpi sample and G) micro-FTIR map of same sample (DEpi) but plotted for intensity of peak at 1110 cm^-1. Bar shows false color scale to indicate intensity with red being highest and blue lowest.</p

Optical images and shrinkage of dried cross sections exposing the lesion depths.

<p>A) demineralized lesion (DE) with nail varnish shown protecting the unexposed surface; B) shrinkage was greatly decreased when demineralized in the present of PI (DEpi) and and was almost undetectable at C) DE-REM; D) DE-REMpi; and E) DEpi-REMpi. Solid red line = original surface location, black dotted line = lesion depth.</p

Average shrinkage measurement.

<p>Average shrinkage measurement.</p

Self-Assembly of Filamentous Amelogenin Requires Calcium and Phosphate: From Dimers via Nanoribbons to Fibrils

Enamel matrix self-assembly has long been suggested as the driving force behind aligned nanofibrous hydroxyapatite formation. We tested if amelogenin, the main enamel matrix protein, can self-assemble into ribbon-like structures in physiologic solutions. Ribbons 17 nm wide were observed to grow several micrometers in length, requiring calcium, phosphate, and pH 4.0–6.0. The pH range suggests that the formation of ion bridges through protonated histidine residues is essential to self-assembly, supported by a statistical analysis of 212 phosphate-binding proteins predicting 12 phosphate-binding histidines. Thermophoretic analysis verified the importance of calcium and phosphate in self-assembly. X-ray scattering characterized amelogenin dimers with dimensions fitting the cross-section of the amelogenin ribbon, leading to the hypothesis that antiparallel dimers are the building blocks of the ribbons. Over 5–7 days, ribbons self-organized into bundles composed of aligned ribbons mimicking the structure of enamel crystallites in enamel rods. These observations confirm reports of filamentous organic components in developing enamel and provide a new model for matrix-templated enamel mineralization

Piezoelectric Tensor of Collagen Fibrils Determined at the Nanoscale

Piezoelectric properties of rat tail tendons, sectioned at angles of 0, 59, and 90° relative to the plane orthogonal to the major axis, were measured using piezoresponse force microscopy. The piezoelectric tensor at the length scale of an individual fibril was determined from angle-dependent in-plane and out-of-plane piezoelectric measurements. The longitudinal piezoelectric coefficient for individual fibrils at the nanoscale was found to be roughly an order of magnitude greater than that reported for macroscopic measurements of tendon, the low response of which stems from the presence of oppositely oriented fibrils, as confirmed here