Self-aligning amelogenin nanoribbons in oil–water system

Abstract

The highly organized microstructure of dental enamel is a result of protein-guided anisotropic growth of apatite nanofibers. It is established that amelogenin proteins, the main constituent of the developing enamel matrix, form nanospheres in vitro, but the amphiphilic nature of the full-length protein conveys the possibility of generating more complex structures as observed with other surfactant-like molecules. This study tested if the use of metastable oil-water emulsions can induce supramolecular assemblies of amelogenin. Recombinant full-length amelogenin, rH174, was mixed into octanol/ethyl acetate preparations of different ratios to form emulsions at pH 4.5 and 7.4. Atomic force and electron microscopy showed the formation of 16.7±1.0 nm wide nanoribbons which grew to several micrometer length over a period of days. Nanoribbons formed from reverse micelles by enabling hydrophobic tails of the molecules to interact while preventing the formation of amelogenin nanospheres. Ribbon formation required the presence of calcium and phosphate ions and may be localized at a dark central line along the amelogenin ribbons. The ribbons have a strong tendency to align in parallel maintaining 5 to 20nm space between each other. The growth rates and number of ribbons were significantly higher at pH 4.5 and related to the metastability of the emulsion. A model for ribbon extension proposes the addition of short segments or amelogenin dimers to the ends of the ribbon. The formation of self-aligning and uniaxially elongating amelogenin structures triggered by the presence of calcium and phosphate may represent a suitable new model for protein controlled mineralization in enamel.

Matching 4.7-Å XRD Spacing in Amelogenin Nanoribbons and Enamel Matrix

“…Our analysis suggests that the nanoribbons are predominantly (75%) comprised of antiparallel beta-sheets, as proposed in a previous model (He et al, 2011;Martinez-Avila et al, 2012). The diffraction pattern at 4.7 Å corresponds very well with observations made by others on the enamel matrix, indicating that this form of assembled structure may also be adopted by amelogenin during amelogenesis.…”

“…The nanoribbon configuration of amelogenin is quite different. Nanoribbons of rH174 have a precise width of 16.7 (± 1) nm, possibly related to the width of an antiparallel amelogenin dimer which may constitute the building block of the ribbons (He et al, 2011;Martinez-Avila et al, 2012). There is evidence in the literature that antiparallel β-sheets have a secondary amide-I peak at increased wavenumbers between 1,680 and 1,715 cm -1 (Haris and Chapman, 1995).…”

Amelogenin and enamel biomimetics

“…4B). 20, 43, 57, 58 It was suggested that the nanochain structures were formed through further association of the amelogenin nanospheres (or association of amelogenin oligomers). 43 The bipolar nature of the molecule can facilitate the formation and/or reorganization of the chain structures.…”

L’émail

“…d'assemblage par dépôt électrolytique (ELD) permet également la préparation de polymères composites par coprécipitation simultanée d'AMEL recombinante (rP172) et du phosphate de calcium minéral en conditions physiologiques [34]. Une fois sécrétés, ces polymères s'assemblent pour donner les nanosphères, qui s'associent entres elles pour former les nanorubans qui vont se déposer sur le cristal en formation [5,33]. Les peptides amélaires purifiés à partir du porc (contenant majoritairement des amélogénines) sont commercialisés et utilisés en clinique depuis une dizaine d'années.…”

“…La biomimétique consiste à reproduire et imiter artificiellement les procédés de la nature dans les organismes vivants. À cet effet, de nombreuses études réalisées à partir d'amélogénine [AMEL] recombinante ont été entreprises chez Escherichia coli (malgré l'absence chez cette bactérie de modifications post-traductionnelles permettant la maturation des protéines synthétisées) [4,33]. Néanmoins, une équipe a ainsi reproduit in vitro la formation de nanorubans d'AMEL recombinante (rH174) dans une émulsion huile-eau de micelles inverses en présence d'ions Ca 2+ et PO 4 3-à pH acide.…”