Proline repeat motif length determines matrix pattern and crystal habit

Amelogenins, the principle proteins of the developing enamel matrix, feature proline-rich triple amino acid repeats (PXX motifs) that increase in length during the course of vertebrate evolution. Objectives: To determine the effect of PXX repeat motif peptide length on HAP crystal formation and matrix organization and to determine its atomic structure. Methods: Three peptides containing 12mer, 24mer and 33mer PXX repeat motifs respectively were chemically synthesized and used for sub-studies. First, peptide solutions were prepared on grids, stained using uranyl-acetate, and used to determine subunit-dimensions of 3D-assemblies via transmission electron microscopy (TEM). Second, peptides were added to HAP crystal growth solutions containing 2.5mM calcium chloride and 1.5mM ammonium phosphate, and were adjusted to pH 8.0 using 1mM ammonium hydroxide. Crystal sizes were then determined using TEM. Third, the atomic structure of the longest peptide was determined by Nuclear Magnetic Resonance (NMR). Results: TEM image analysis of PXX matrix structure revealed subunit compartments of 278+/-40 (12mer), 652+/-74 (24mer), and 1190+/-128 (33mer) particles per square micrometer (ppmm) compared to 568+/-79 ppmm (full-length amelogenin). This increase in ppmm resulted in a decrease in subunit compartment size. Dimensions of newly formed apatite crystals grown in PXX peptide solutions were 21.6+/-6.5nm (12mer), 43.0+/-9.9nm (24mer), and 99.6+/-44.3 (33mer) compared to 106.2+/-19.3nm (amelogenin). Differences in matrix particle concentration and crystal sizes were compared using ANOVA and were highly significant. NMR analysis of the 33mer PXX peptide in solution resolved an “S” shape structure allowing for the spacing of apatite crystals. Conclusion: Increased length of the proline-rich amelogenin PXX repeats resulted in significantly reduced size of matrix subunit compartments and increased HAP crystal length. Elongated PXX-repeat motifs as they have evolved in vertebrates might be correlated with increased enamel crystal c-axis dimensions. Funding by NIDCR grant DE13378 to TGHD is gratefully acknowledged.