A solution NMR investigation into the early events of amelogenin nanosphere self-assembly initiated with sodium chloride or calcium chloride.

Dental enamel, the layer of elongated carbonated hydroxayapatite on the outer layer of the tooth, is the hardest tissue in the human body (1). It needs to be strong because it is exposed to repeated masticatory, parafunctional, and occasional impact loading, but unlike the dominant biomineral in the human body (2), mesenchyme-derived bone, enamel cannot self-repair nor can it undergo remodeling, and therefore, it must last a lifetime. Enamel’s resistance to wear and deformation are due to a combination of high mineral content and unique three-dimensional structural organization (3). Ninety-five percent of mature enamel consists of long and narrow crystals of carbonated hydroxyapatite packed into parallel arrays, called enamel rods, that are intricately interwoven into an unique lattice architecture (4, 5). There is little, if any, matrix protein remaining in mature enamel, another feature differentiating enamel markedly from dentin and bone. The nucleation, growth, and organization of enamel (amelogenesis) are extracellular processes orchestrated by one predominant enamel matrix protein, amelogenin (5–8), that is secreted by the inner enamel epithelium (ameloblasts) and found exclusively in the region of newly formed enamel (9). Specific mutations to amelogenin and amelogenin deficient mice both result in dramatic enamel phenotypes similar to amelogenesis imperfecta in humans (5), observations that strongly suggest amelogenin plays an essential role in amelogenesis. The amino- and carboxy terminal regions of this low-molecular-weight (~20 kDa, depending on species) protein are highly conserved among species, suggesting these regions serve important functional roles in mediating enamel biomineralization (10). Except for the hydrophilic and charged C-terminal region (11), amelogenin is hydrophobic in nature (10) and contains a central region enriched in the amino acids P, L, H, and Q (HQP-rich region). Indeed, it is variations in the length of this central HQP-rich region that largely account for the differences in the size of amelogenin among various species. The secondary structure of amelogenin has been studied extensively by circular dichroism (CD 1) (12–14) and small-angle X-ray spectroscopy (SAXS) (15). The general consensus is that in monomeric form, the tyrosine-rich N-terminal region forms an extended β-sheet/β-strand structure, while the HQP-rich region adopts aβ-spiral/polyproline II (PPII) structure (5, 14,15). This structure appears to be very sensitive to pH, temperature, ionic strength, and metal ions. Of the many interesting features of amelogenin, perhaps the most remarkable is its ability, under the correct conditions, to self-assemble into a unique quaternary structure, termed nanosphere, that appears to be an essential functional and structural component of the secretory-stage enamel matrix (8, 16, 17). Amelogenin nanospheres composed of multiple amelogenin molecules are 20–50 nm in radius and have been observed both in vivo (8) and in vitro (7). In vitro, amelogenin molecules will spontaneously self-associate to form nanospheres under many different types of conditions(17–19). Mutants of amelogenin that have disrupted nanosphere formation in vitro (20) are observed to cause malformed enamel in knock-in mice in vivo (21) supporting the importance of this quaternary structure in enamel biomineralization. Protein proteolysis studies (22) and yeast two-hybrid assays (23) suggest that the regions of the protein essential in nanosphere self-assembly are M1–M42 and S157–K173. It is hypothesized that nanosphere assembly occurs through the progressive accretion of amelogenin molecules with monomers first associating into dimer and trimer building blocks (19, 24). However, molecular details of the self-assembly process are still not well understood. This is partially because amelogenin self-association is a complex process and depends on the interplay between protein concentration and the many properties of the solution (ionic strength, pH, solutes, and temperature) (17–19) that, in turn, affect the physical properties of the protein (hydrophobicity and electrostatics) (5, 25, 26). To better understand details of the initial events of nanosphere self-assembly, we have used solution NMR spectroscopy and dynamic light scattering to probe the behavior of mouse amelogenin (11), at a fixed concentration, as a function of increasing ionic strength using the salts sodium chloride and calcium chloride. The smaller monomeric (~20 kDa) and dimeric (~40 kDa) species are accessible to solution state NMR experiments unlike the much larger, fully assembled, nanospheres (~2,000 kDa). Because we have assigned the 1H, 13C, and 15N chemical shifts for amelogenin under acidic conditions where it is a monomer in solution (27), it is possible to identify the specific residues involved in the early stages of nanosphere assembly by following perturbations to their amide chemical shifts in the 1H–15N HSQC spectrum as a function of salt concentration. Changes in the chemical environment of the nuclei at interaction interfaces are often accompanied by perturbations, typically chemical shift and/or intensity reductions to the backbone 1HN and 15N resonances (28, 29). In this instance, the major changes observed in the 1H–15N HSQC spectra were the disappearance of cross-peaks, an observation that indicates a change in dynamics at or near the affected residues into a regime of intermediate motion (microsecond to millisecond) that is unfavorable in the NMR time scale (28, 30).