The onset of amelogenin nanosphere aggregation studied by small-angle X-ray scattering and dynamic light scattering
Introduction
Nature has developed a very successful strategy to design mechanically resistant materials, such as vertebrate bones and teeth, and the mollusk shell, by using minerals for the reinforcement of organic tissues. Their complex mineralized structures, consisting of multiple hierarchical levels of organization, are crucial factors that sustain the mechanical properties of these protein/mineral composites (Gao et al., 2003, Nalla et al., 2003, Weiner and Wagner, 1998). In teeth, a relatively soft but tough core made from dentin and a hard outer layer consisting of enamel combine to withstand abrasion as well as fractures during mastication. Enamel provides teeth with the required wear-resistance. A high mineral content (>95% by weight) ensures high hardness and a well-organized structure, consisting of interwoven bundles of rod-like hydroxyapatite (HAP) crystals, allows for a sufficient increase of toughness in comparison with the very brittle pure mineral phase. In contrast to bone, which is able to repair arising defects, the enamel structure, once formed, must fulfill its function for a lifetime without any further remodeling. This astonishing performance strongly motivates an investigation of the mechanisms by which such a durable structure is built.
Enamel formation begins within the developing enamel organ as specialized cells differentiate to form ameloblasts. As layers of these cells retreat from the dentino-enamel junction, they secrete enamel matrix proteins that serve to regulate the almost immediate nucleation and growth of parallel arrays of long and very thin ribbons of enamel mineral (Cuisinier et al., 1992, Daculsi and Kerebel, 1978). Thus, unlike other vertebrate tissues, enamel mineral does not form within a preformed matrix (Fincham et al., 1999). Also, unlike other mineralized tissues, the enamel matrix is progressively removed as the biomineral matures, leading to the formation of the highly mineralized enamel tissue. Amelogenin is the most abundant protein in this secretory-stage enamel matrix. It mainly contains hydrophobic domains, with the exception of hydrophilic N- and C-terminals (Zeichner-David, 2001). As mineral crystals grow within the protein matrix, amelogenin is successively degraded (Bartlett et al., 1998, Moradian-Oldak et al., 1994, Moradian-Oldak et al., 1998a, Simmer et al., 1998). Soon after the secretion of the protein, the hydrophilic carboxy-terminal is cleaved. Importantly, the full-length amelogenin is localized exclusively in the region of newly formed mineral (Uchida et al., 1991), thus suggesting a unique role of the full-length molecule during enamel formation. Although other enamel matrix proteins are likely to play critical roles in enamel mineral formation (Fincham et al., 1999), the critical importance of amelogenin is supported by recent studies that have shown that the amelogenin-null mouse exhibits a dramatic enamel phenotype (Gibson et al., 2001).
The self-assembly of amelogenin into nanospheres may play a key role in the biomineralization of enamel. A number of studies have shown that native and recombinant amelogenins aggregate to form mono-dispersed particles in the nanometer size range (e.g., Fincham et al., 1994, Fincham et al., 1995, Moradian-Oldak et al., 1998b, Moradian-Oldak et al., 2000) in vitro. It was suggested that amelogenins self-assemble to form organized supramolecular structures that may facilitate crystal organization. Such suggestions are based, in part, on the detection of chains of nanometer sized spheres in vivo by TEM examination of sections of forming enamel (Bai and Warshawsky, 1985, Fincham et al., 1995, Robinson et al., 1981, Smales, 1975). Rows of amelogenin nanospheres, separating the mineral crystals have been postulated as the main structural components in the developing enamel matrix (Fincham et al., 1995, Robinson et al., 1981). It has been suggested that such aggregates alone or in combination with other proteins may facilitate the nucleation and organization of mineral phases (Fincham et al., 1999). Throughout the last decade, the self-assembly of amelogenin at the nanoscale level as well as the effects of amelogenins on the behavior of crystal growth have been extensively studied (reviewed in Moradian-Oldak, 2001; recently Beniash et al., 2005, Iijima and Moradian-Oldak, 2004, Iijima and Moradian-Oldak, 2005). On the microscale level, the structure of the organic enamel matrix is controlled in part by the migration of ameloblast cells (Paine et al., 2001). However, some details of the assembly process of amelogenin are not yet fully understood, especially the potential interactions between the protein nanospheres. Further studies are needed to investigate the possible formation of an ordered array consisting of multiple amelogenin nanospheres, which could ultimately serve as a suitable template for biomineralization and the fabrication of the highly ordered enamel tissue.
In the present paper, we have studied the temperature and pH-dependent self-assembly of two recombinant murine amelogenins by means of dynamic light scattering (DLS) and small-angle X-ray and neutron scattering (SAXS and SANS). These methods are sensitive to totally different features and hence, in combination, provide unique insight into the structure of protein aggregates. DLS monitors diffusion and allows for an indirect estimation of the size of the moving particles. In contrast, SAXS directly reflects structural features related to electron-density differences in materials (reviewed in Fratzl, 2003). In addition to the full-length recombinant protein rM179, we investigated an engineered version lacking a 13 amino acid hydrophilic C-terminus (rM166), to provide insight into the role of this protein segment in amelogenin self-assembly. The aim of our investigation is to elucidate how amelogenin nanospheres interact to form a matrix, which is able to direct the growth of mineral crystals in developing enamel.
Section snippets
Recombinant protein preparation
Recombinant mouse amelogenins, rM179 and rM166, were produced in bacteria and purified, as previously described (Simmer et al., 1994). As indicated in Fig. 1, the recombinant proteins differ from their native counterparts by lacking a single phosphate group at Ser16 and the N-terminal methionine. Thus, rM179 represents the full-length recombinant amelogenin, while rM166 is a major proteolytic cleavage product of the full-length mouse amelogenin lacking the 13 amino acid hydrophilic C-terminal
Results
Recombinant proteins rM166 and rM179 were investigated in solution at different pH and temperatures. The behavior of both proteins at acidic pH differed significantly from that observed at pH 8. Fig. 2 shows the comparison of the SAXS signals of rM179 at 20 °C for two different pH values. Under acidic conditions (pH < 3.5) we observed a very weak scattering signal as a function of the scattering vector Q, with a slope of approximately −1, indicating an elongated shape of the protein particles.
Discussion
The comparison between the size of the nanospheres, as measured by SAXS and DLS, leads to two major surprises. First, the nanospheres appear much smaller in SAXS than in DLS at all temperatures and, second, one observes a dramatic size increase at high temperatures by DLS, which is not visible in the SAXS data. The explanation must reside in the different physical basis of the two measurements, DLS and SAXS. The hydrodynamic radius, determined by DLS, probes the diffusion characteristics of the
Acknowledgments
We are grateful to Dr. Richard Görgl and Mag. Günther Maier for their help with high resolution laboratory SAXS measurements. The authors thank Dr. Roland May for his support during the neutron scattering experiments at the Institut Laue-Langevin, instrument D22. This work was supported in part by a Grant PO1-DE-13237 (H.M.) from the National Institute of Dental and Craniofacial Research.
References (44)
- et al.
Selective adsorption of porcine-amelogenins onto hydroxyapatite and their inhibitory activity on hydroxyapatite growth in supersaturated solutions
Calcif. Tissue Int.
(1987) - et al.
Morphological-studies on the distribution of enamel matrix proteins using routine electron-microscopy and freeze-fracture replicas in the rat incisor
Anat. Rec.
(1985) - et al.
Enamelysin mRNA displays a developmentally defined pattern of expression and encodes a protein which degrades amelogenin
Connect. Tissue Res.
(1998) - et al.
Absolute Rayleigh ratios of four solvents at 488 nm
Macromolecules
(1986) - et al.
The effect of recombinant mouse amelogenins on the formation and organization of hydroxyapatite crystals in vitro
J. Struct. Biol.
(2005) - et al.
Information on polydispersity and branching from combined quasi-elastic and integrated scattering
Macromolecules
(1980) - et al.
Higher-order organization by mesoscale self-assembly and transformation of hybrid nanostructures
Angew. Chem. Int. Ed.
(2003) - et al.
Human amelogenesis. 1 high resolution electron microscope study of ribbon-like crystals
Calcif. Tissue Int.
(1992) - et al.
High resolution electron microscope study of human enamel crystallites: size, shape and growth
J. Ultrastruct. Res.
(1978) - et al.
Supramolecular assembly of amelogenin nanospheres into birefringent microribbons
Science
(2005)
Self-assembly of a recombinant amelogenin protein generates supramolecular structures
J. Struct. Biol.
Evidence for amelogenin “Nanospheres” as functional components of secretory-stage enamel matrix
J. Struct. Biol.
The structural biology of the developing dental enamel matrix
J. Struct. Biol.
Small-angle scattering in materials science, a short review of applications in alloys, ceramics and composite materials
J. Appl. Cryst.
Materials become insensitive to flaws at nanoscale: lessons from nature
Proc. Natl. Acad. Sci. USA
Amelogenin-deficient mice display an amelogenesis imperfecta phenotype
J. Biol. Chem.
Small-Angle Scattering of X-rays
Some geometrical factors in light scattering apparatus
J. Opt. Soc. Am.
Effects of bovine amelogenins on the crystal morphology of octacalcium phosphate in a model system of tooth enamel formation
J. Cryst. Growth
Elongated growth of octacalcium phosphate crystals in recombinant amelogenin gels under controlled ionic flow
J. Dent. Res.
Control of octacalcium phosphate and apatite crystal growth by amelogenin matrices
J. Mater. Chem.
Control of apatite crystal growth in a fluoride containing amelogenin-rich matrix
Biomaterials
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