Structural adaptation of tooth enamel protein amelogenin in the presence of SDS micelles

Amelogenin, the major extracellular matrix protein of developing tooth enamel is intrinsically disordered. Through its interaction with other proteins and mineral, amelogenin assists enamel biomineralization by controlling the formation of highly organized enamel crystal arrays. We used circular dichroism (CD), dynamic light scattering (DLS), fluorescence, and NMR spectroscopy to investigate the folding propensity of recombinant porcine amelogenin rP172 following its interaction with SDS, at levels above critical micelle concentration. The rP172‐SDS complex formation was confirmed by DLS, while an increase in the structure moiety of rP172 was noted through CD and fluorescence experiments. Fluorescence quenching analyses performed on several rP172 mutants where all but one Trp was replaced by Tyr at different sequence regions confirmed that the interaction of amelogenin with SDS micelles occurs via the N‐terminal region close to Trp25 where helical segments can be detected by NMR. NMR spectroscopy and structural refinement calculations using CS‐Rosetta modeling confirm that the highly conserved N‐terminal domain is prone to form helical structure when bound to SDS micelles. Our findings reported here reveal interactions leading to significant changes in the secondary structure of rP172 upon treatment with SDS. These interactions may reflect the physiological relevance of the flexible nature of amelogenin and its sequence specific helical propensity that might enable it to structurally adapt with charged and potential targets such as cell surface, mineral, and other proteins during enamel biomineralization. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 525–535, 2014.     

Interactions of amelogenin with phospholipids

Amelogenin protein has the potential to interact with other enamel matrix proteins, mineral, and cell surfaces. We investigated the interactions of recombinant amelogenin rP172 with small unilamellar vesicles as model membranes, toward the goal of understanding the mechanisms of amelogenin–cell interactions during amelogenesis. Dynamic light scattering (DLS), fluorescence spectroscopy, circular dichroism (CD), and nuclear magnetic resonance (NMR) were used. In the presence of phospholipid vesicles, a blue shift in the Trp fluorescence emission maxima of rP172 was observed (～334 nm) and the Trp residues of rP172 were inaccessible to the aqueous quencher acrylamide. DLS studies indicated complexation of rP172 and phospholipids, although the possibility of fusion of phospholipids following amelogenin addition cannot be ruled out. NMR and CD studies revealed a disorder–order transition of rP172 in a model membrane environment. Strong fluorescence resonance energy transfer from Trp in rP172 to DNS‐bound‐phospholipid was observed, and fluorescence polarization studies indicated that rP172 interacted with the hydrophobic core region of model membranes. Our data suggest that amelogenin has ability to interact with phospholipids and that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 96–108, 2015.     

Dissecting amelogenin protein nanospheres: characterization of metastable oligomers

Amelogenin self-assembles to form an extracellular protein matrix, which serves as a template for the continuously growing enamel apatite crystals. To gain further insight into the molecular mechanism of amelogenin nanosphere formation, we manipulated the interactions between amelogenin monomers by altering pH, temperature, and protein concentration to create isolated metastable amelogenin oligomers. Recombinant porcine amelogenins (rP172 and rP148) and three different mutants containing only a single tryptophan (Trp(161), Trp(45), and Trp(25)) were used. Dynamic light scattering and fluorescence studies demonstrated that oligomers were metastable and in constant equilibrium with monomers. Stable oligomers with an average hydrodynamic radius (R(H)) of 7.5 nm were observed at pH 5.5 between 4 and 10 mg · ml(-1). We did not find any evidence of a significant increase in folding upon self-association of the monomers into oligomers, indicating that they are disordered. Fluorescence experiments with single tryptophan amelogenins revealed that upon oligomerization the C terminus of amelogenin (around residue Trp(161)) is exposed at the surface of the oligomers, whereas the N-terminal region around Trp(25) and Trp(45) is involved in protein-protein interaction. The truncated rP148 formed similar but smaller oligomers, suggesting that the C terminus is not critical for amelogenin oligomerization. We propose a model for nanosphere formation via oligomers, and we predict that nanospheres will break up to form oligomers in mildly acidic environments via histidine protonation. We further suggest that oligomeric structures might be functional components during maturation of enamel apatite.     

Probing the self-association, intermolecular contacts, and folding propensity of amelogenin

Amelogenins are an intrinsically disordered protein family that plays a major role in the development of tooth enamel, one of the most highly mineralized materials in nature. Monomeric porcine amelogenin possesses random coil and residual secondary structures, but it is not known which sequence regions would be conformationally attractive to potential enamel matrix targets such as other amelogenins (self-assembly), other matrix proteins, cell surfaces, or biominerals. To address this further, we investigated recombinant porcine amelogenin (rP172) using "solvent engineering" techniques to simultaneously promote native-like structure and induce amelogenin oligomerization in a manner that allows identification of intermolecular contacts between amelogenin molecules. We discovered that in the presence of 2,2,2-trifluoroethanol (TFE) significant folding transitions and stabilization occurred primarily within the N- and C-termini, while the polyproline Type II central domain was largely resistant to conformational transitions. Seven Pro residues (P2, P127, P130, P139, P154, P157, P162) exhibited conformational response to TFE, and this indicates these Pro residues act as folding enhancers in rP172. The remaining Pro residues resisted TFE perturbations and thus act as conformational stabilizers. We also noted that TFE induced rP172 self-association via the formation of intermolecular contacts involving P4-H6, V19-P33, and E40-T58 regions of the N-terminus. Collectively, these results confirm that the N- and C-termini of amelogenin are conformationally responsive and represent potential interactive sites for amelogenin-target interactions during enamel matrix mineralization. Conversely, the Pro, Gln central domain is resistant to folding and this may have important functional significance for amelogenin.     

The role of secondary structure in the entropically driven amelogenin self-assembly

Amelogenin, the major extracellular enamel matrix protein, plays critical roles in controlling enamel mineralization. This generally hydrophobic protein self-assembles to form nanosphere structures under certain solution conditions. To gain clearer insight into the mechanisms of amelogenin self-assembly, we first investigated the occurrences of secondary structures within its sequence. By applying isothermal titration calorimetry (ITC), we determined the thermodynamic parameters associated with protein-protein interactions and with conformational changes during self-assembly. The recombinant porcine full length (rP172) and a truncated amelogenin lacking the hydrophilic C-terminal (rP148) were used. Circular dichroism (CD) measurements performed at low concentrations (<5 microM) revealed the presence of the polyproline-type II (PPII) conformation in both amelogenins in addition to alpha-helix and unordered conformations. Structural transition from PPII/unordered to beta-sheet was observed for both proteins at higher concentrations (>62.5 microM) and upon self-assembly. ITC measurements indicated that the self-assembly of rP172 and rP148 is entropically driven (+DeltaS(A)) and energetically favorable (-DeltaG(A)). The magnitude of enthalpy (DeltaH(A)) and entropy changes of assembly (DeltaS(A)) were smaller for rP148 than rP172, whereas the Gibbs free energy change of assembly (DeltaG(A)) was not significantly different. It was found that rP172 had higher PPII content than rP148, and the monomer-multimer equilibrium for rP172 was observed in a narrower protein concentration range when compared to rP148. The large positive enthalpy and entropy changes in both cases are attributed to the release of ordered water molecules and the associated entropy gain (due to the hydrophobic effect). These findings suggest that PPII conformation plays an important role in amelogenin self-assembly and that rP172 assembly is more favorable than rP148. The data are direct evidence for the notion that hydrophobic interactions are the main driving force for amelogenin self-assembly.     

Effects of phosphorylation on the self-assembly of native full-length porcine amelogenin and its regulation of calcium phosphate formation in vitro

The self-assembly of the predominant extracellular enamel matrix protein amelogenin plays an essential role in regulating the growth and organization of enamel mineral during early stages of dental enamel formation. The present study describes the effect of the phosphorylation of a single site on the full-length native porcine amelogenin P173 on self-assembly and on the regulation of spontaneous calcium phosphate formation in vitro. Studies were also conducted using recombinant non-phosphorylated (rP172) porcine amelogenin, along with the most abundant amelogenin cleavage product (P148) and its recombinant form (rP147). Amelogenin self-assembly was assessed using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Using these approaches, we have shown that self-assembly of each amelogenin is very sensitive to pH and appears to be affected by both hydrophilic and hydrophobic interactions. Furthermore, our results suggest that the phosphorylation of the full-length porcine amelogenin P173 has a small but potentially important effect on its higher-order self-assembly into chain-like structures under physiological conditions of pH, temperature, and ionic strength. Although phosphorylation has a subtle effect on the higher-order assembly of full-length amelogenin, native phosphorylated P173 was found to stabilize amorphous calcium phosphate for extended periods of time, in sharp contrast to previous findings using non-phosphorylated rP172. The biological relevance of these findings is discussed.     

Interaction between the enamel matrix proteins amelogenin and ameloblastin

Enamel matrix consists of amelogenin and non-amelogenins. Though amelogenin is not involved in nucleation of minerals, the enamel mineralization is impaired when amelogenin or other matrix protein (ameloblastin/enamelin) genes are mutated. We hypothesize that amelogenin may promote enamel mineralization by interacting with the calcium-binding matrix proteins. Specific binding of amelogenin to N-acetylglucosamine (GlcNAc), GlcNAc-mimicking peptides (GMps), and their carrier proteins and the identification of amelogenin-trityrosyl-motif-peptide (ATMP) as a GlcNAc/GMp-binding domain in amelogenin favor the hypothesis. This study tested the interaction of amelogenin with ameloblastin, a carrier of GMp sequence at intermittent sites. Neither GlcNAc nor sialic acids were identified in the recombinant-ameloblastin. Amelogenin bound to recombinant-ameloblastin in both Western blots and in ELISA. More specifically, [(3)H]ATMP bound to both recombinant and native ameloblastins. Dosimetry and Scatchard analyses showed the specific interaction between ATMP and ameloblastin, suggesting that amelogenin may interact with ameloblastin to form a heteromolecular assembly.     

Morphological and immunocytochemical analyses on the effects of diet-induced hypocalcemia on enamel maturation in the rat incisor.

During the maturation stage of amelogenesis, the loss of matrix proteins combined with an accentuated but regulated influx of calcium and phosphate ions into the enamel layer results in the "hardest" tissue of the body. The aim of the present investigation was to examine the effects of chronic hypocalcemia on the maturation of enamel. Twenty-one-day old male Wistar rats were given a calcium-free diet and deionized water for 28 days, while control animals received a normal chow. The rats were perfused with aldehyde and the mandibular incisors were processed for histochemical and ultrastructural analyses and for postembedding colloidal gold immunolabeling with antibodies to amelogenin, ameloblastin, and albumin. The maturation stage enamel organ in hypocalcemic rats exhibited areas with an apparent increase in cell number and the presence of cyst-like structures. In both cases the cells expressed signals for ameloblastin and amelogenin. The content of the cysts was periodic acid-Schiff- and periodic acid-silver nitrate-methanamine-positive and immunolabeled for amelogenin, ameloblastin, and albumin. Masses of a similar material were also found at the enamel surface in depressions of the ameloblast layer. In addition, there were accumulations of glycoproteinaceous matrix at the interface between ameloblasts and enamel. In decalcified specimens, the superficial portion of the enamel matrix sometimes exhibited the presence of tubular crystal "ghosts." The basal lamina, normally separating ameloblasts and enamel during the maturation stage, was missing in some areas. Enamel crystals extended within membrane invaginations at the apical surface of ameloblasts in these areas. Immunolabeling for amelogenin, ameloblastin, and albumin over enamel was variable and showed a heterogeneous distribution. In contrast, enamel in control rats exhibited a homogeneous labeling for amelogenin, a concentration of ameloblastin at the surface, and weak reactivity for albumin. These results suggest that diet-induced chronic hypocalcemia interferes with both cellular and extracellular events during enamel maturation.     

pH triggered self-assembly of native and recombinant amelogenins under physiological pH and temperature in vitro

Self-assembly of the extracellular matrix protein amelogenin is believed to play an essential role in regulating the growth and organization of enamel crystals during enamel formation. This study examines the effect of temperature and pH on amelogenin self-assembly under physiological pH conditions in vitro, using dynamic light scattering, turbidity measurements, and transmission electron microscopy. Full-length recombinant amelogenins from mouse (rM179) and pig (rP172) were investigated, along with proteolytic cleavage products (rM166 and native P148) lacking the hydrophilic C-terminus of parent molecules. Results indicated that the self-assembly of full-length amelogenin is primarily triggered by pH in the temperature range from 13 to 37 degrees C and not by temperature. Furthermore, very large assemblies of all proteins studied formed through the rearrangement of similarly sized nanospherical particles, although at different pH values: pH 7.7 (P148), pH 7.5 (rM166), pH 7.2 (rP172), and pH 7.2 (rM179). Structural differences were also observed. The full-length molecules formed apparently tightly connected elongated, high-aspect ratio assemblies comprised of small spheres, while the amelogenin cleavage products appeared as loosely associated spherical particles, suggesting that the hydrophilic C-terminus plays an essential role in higher-order amelogenin assembly. Hence, tightly controlled pH values during secretory amelogenesis may serve to regulate the functions of both full-length and cleaved amelogenins.     

Altering biomineralization by protein design

To create a bioceramic with unique materials properties, biomineralization exploits cells to create a tissue-specific protein matrix to control the crystal habit, timing, and position of the mineral phase. The biomineralized covering of vertebrate teeth is enamel, a distinctive tissue of ectodermal origin that is collagen-free. In forming enamel, amelogenin is the abundant protein that undergoes self-assembly to contribute to a matrix that guides its own replacement by mineral. Conserved domains in amelogenin suggest their importance to biomineralization. We used gene targeting in mice to replace native amelogenin with one of two engineered amelogenins. Replacement changed enamel organization by altering protein-to-crystallite interactions and crystallite stacking while diminishing the ability of the ameloblast to interact with the matrix. These data demonstrate that ameloblasts must continuously interact with the developing matrix to provide amelogenin-specific protein to protein, protein to mineral, and protein to membrane interactions critical to biomineralization and enamel architecture while suggesting that mutations within conserved amelogenin domains could account for enamel variations preserved in the fossil record.     

Reduced hydrolysis of amelogenin may result in X-linked amelogenesis imperfecta

Amelogenesis imperfecta (AI) is a group of inherited disorders with defective tooth enamel formation caused by various gene mutations. One of the mutations substitutes a cytidine to adenine in exon 6 of the X-chromosomal amelogenin gene, which results in a proline to threonine change in the expressed amelogenin. This transformation is four amino acids N terminal to the proteinase cleavage site in amelogenin for enamel matrix metalloproteinase-20 (MMP-20), also known as enamelysin. MMP-20 effects the release of tyrosine rich amelogenin peptide (TRAP) from amelogenin. This study evaluated the rate MMP-20 hydrolyzes the putative mutated amelogenin cleavage site. The proteolytic site was modeled as a substrate by two synthetic peptides, P1 (SYGYEPMGGWLHHQ) and M1 (SYGYETMGGWLHHQ), selected from residue 36-49 of the amino acid sequence for amelogenin and the respective X-linked amelogenin mutant. Recombinant metalloproteinase-20 (rMMP-20) was used to digest the oligopeptides and the truncated peptides were separated by reversed phase HPLC and identified by mass spectrometry. The results demonstrate that both peptides are cleaved between tryptophan and leucine, matching the TRAP cutting site found in tooth enamel. However, the apparent first order rate of digestion of the mutation containing peptide by rMMP-20 was approximately 25 times slower than that of the non-mutated peptide. This study suggests that the reduced rate of TRAP formation due to a single amino acid substitution may alter enamel formation and consequently result in amelogenesis imperfecta.     

Analysis of secondary structure and self‐assembly of amelogenin by variable temperature circular dichroism and isothermal titration calorimetry

Amelogenin is a proline‐rich enamel matrix protein known to play an important role in the oriented growth of enamel crystals. Amelogenin self‐assembles to form nanospheres and higher order structures mediated by hydrophobic interactions. This study aims to obtain a better insight into the relationship between primary–secondary structure and self‐assembly of amelogenin by applying computational and biophysical methods. Variable temperature circular dichroism studies indicated that under physiological pH recombinant full‐length porcine amelogenin contains unordered structures in equilibrium with polyproline type II (PPII) structure, the latter being more populated at lower temperatures. Increasing the concentration of rP172 resulted in the promotion of folding to an ordered β‐structured assembly. Isothermal titration calorimetry dilution studies revealed that at all temperatures, self‐assembly is entropically driven due to the hydrophobic effect and the molar heat of assembly (ΔH_A) decreases with temperature. Using a computational approach, a profile of domains in the amino acid sequence that have a high propensity to assemble and to have PPII structures has been identified. We conclude that the assembly properties of amelogenin are due to complementarity between the hydrophobic and PPII helix prone regions. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Spatiotemporal expression of ameloblastin isoforms during murine tooth development

Ameloblasts synthesize and secrete the enamel matrix proteins (amelogenin, ameloblastin, and enamelin). This investigation examined the profiles of ameloblastin in the ameloblasts and in the enamel matrix during different postnatal (PN) days (days 0-9) of development of mouse molar, using an antibody specific for C-terminal sequence of ameloblastin (Ct; GNKVHQPQVHNAWRF). Ameloblastin is found in three different molecular sizes (37, 55, and 66 kDa) in both ameloblasts and enamel matrix during PN development. In the ameloblasts, the sequence of expression of these fractions varied. The 37-kDa fraction was observed (even before the appearances of mRNA of the proteases, enamelysin and kallikrein-4) on days 0 and 1, persisted until day 3, and was not found thereafter. Other isoforms (55 and 66 kDa) distinctly appeared in ameloblasts after day 1, reached a peak on day 5, and remained thereafter. The Ct-positive granules appeared beaded in the ameloblasts on day 3. In the extracellular matrix, a 37-kDa (but not 66- or 55-kDa) fraction was detected on days 0 and 1 and remained in the matrix throughout the PN days. The larger isoforms (55 and 66 kDa) appeared in the enamel matrix from day 3 onward. On days 0-3, but not later, the 37-kDa isoform co-localizes with amelogenin in Tomes' process and formative enamel, as revealed by laser scan confocal microscopy. Autoradiography confirmed accumulation of 3H-labeled amelogenin trityrosyl motif peptide in the region of Tomes' process and formative enamel from day 0 to 3. These observations suggest that the 37-kDa isoform interacts with amelogenin during early tooth development.     

Amelogenin "nanorods" formation during proteolysis by Mmp-20

Amelogenin is cleaved by enamelysin (Mmp-20) soon after its secretion, and the cleavage products accumulate in specific locations during enamel formation, suggesting that parent amelogenin proteolysis is necessary for activating its functions. To investigate the precise roles of Mmp-20 and its influence on the assembly of amelogenin, an in vitro enzymatic digestion process mimicking the initial stages of amelogenin proteolysis was investigated at near-physiological conditions using recombinant porcine amelogenin (rP172) and enamelysin. Hierarchically organized nanorod structures formed during different digestion stages were detected by TEM. At the earliest stage, uniformly dispersed parent amelogenin spherical particles, mixed with some darker stained smaller spheres, and accompanying elongated chain-like nanostructures were observed. Cylindrical nanorods, which appeared to be the result of tight assembly of thin subunit cylindrical discs with thicknesses ranging from ∼2.5 to ∼6.0 nm, were formed after an hour of proteolysis. These subunit building blocks stacked to form nanorods with maximum length of ∼100 nm. With the production of more cleavage products, additional morphologies spontaneously evolved from the cylindrical nanorods. Larger ball-like aggregates ultimately formed at the end of proteolysis. The uniform spherical particles, nanorods, morphological patterns evolved from nanorods, and globular aggregated microstructures were successively formed by means of co-assembly of amelogenin and its cleavage products during a comparatively slow proteolysis process. We propose that, following the C-terminal cleavage of amelogenin, co-assembly with its fragments leads to formation of nanorod structures whose properties eventually dictate the super-structural organization of enamel matrix, controlling the elongated growth of enamel apatite crystals.     

A transgenic animal model resembling amelogenesis imperfecta related to ameloblastin overexpression

Genetic diseases that affect tooth enamel are grouped under the classification amelogenesis imperfecta. Human pedigrees and experiments on transgenic and null mice have all demonstrated that mutations to the secreted proteins amelogenin, enamelin, and enamelysin result in visibly, structurally, or mechanically defective enamel. In an attempt to better define a physiologic function for ameloblastin during enamel formation, we have produced transgenic mice that misexpress the ameloblastin gene. These transgenic animals exhibit imperfections in their enamel that is evident at the nanoscale level. Specifically, ameloblastin overexpression influences enamel crystallite habit and enamel rod morphology. These findings suggest enamel crystallite habit and rod morphology are influenced by the temporal and spatial expression of ameloblastin and may implicate the role of the ameloblastin gene locus in the etiology of a number of undiagnosed autosomally dominant cases of amelogenesis imperfecta.     

Amelogenin sequence and enamel biomineralization in Rana pipiens

The amelogenin gene contributes the majority of tooth enamel proteins and plays a significant role in enamel biomineralization. While several mammalian and reptilian amelogenins have been cloned and sequenced, basal vertebrate amelogenin evolution remains to be understood. In order to start elucidating the structure and function of amelogenins in the evolution of enamel, the leopard frog (Rana pipiens) was used as a model. Tissues from Rana pipiens teeth were analyzed for enamel structure and RNA extracts were processed for sequence analysis. Electron microscopy revealed that Rana pipiens enamel contains long and parallel crystals similar to mammalian enamel, while immunoreactions confirmed the site-specific localization of cross-reactive amelogenins in Rana pipiens enamel. Sequencing of amelogenin PCR products revealed a 782bp cDNA with a 546-nucleotide coding sequence encoding 181 amino acids. The homology of the newly discovered Rana pipiens amelogenin nucleotide and amino acid sequence with the published mouse amelogenin was 38.6% and 45%, respectively. These findings report the first complete amelogenin cDNA sequence in amphibians and indicate a close homology between mammalian enamel formation and Rana pipiens enamel biomineralization.     

Expression of ameloblastin during enamel formation in a crocodile

Ameloblastin is an enamel-specific protein that plays critical roles in enamel formation, as well as adhesion between ameloblasts and the enamel matrix, as shown by analyses of ameloblastin-null mice. In the present study, we produced two distinct antibodies that recognize the N-terminus and C-terminus regions of caiman ameloblastin, in order to elucidate the fate of ameloblastin peptides during tooth development. An immunohistochemical study using the antibodies showed that caiman ameloblastin was a tooth-specific matrix protein that may initially be cleaved into two groups, N- and C-terminal peptides, as shown in mammals. The distribution of the N-terminal peptides was much different from that of the C-terminal peptides during enamel formation; however, it was similar to that of mammalian ameloblastin. Although ameloblastin is thought to have a relationship with the enamel prismatic structure in mammals, in the caiman, which has non-prismatic enamel, functional ameloblastin has no relationship with any enamel structure. Consequently, it is suggested that ameloblastin has kept its original functions during the evolutionary transition from reptiles to mammals and that it has been conserved in both lineages during more than 200 million years of evolution. Our results support the notion that ameloblastin acts as a factor for ameloblast adhesion to enamel matrix, because distribution of the C-terminal peptides was consistently restricted on the surface layers of enamel matrix specimens ranging from immature to nearly completely mature. The principal molecules that provide the adhesive function are presumably C-terminal peptides.     

Altered amelogenin self-assembly based on mutations observed in human X-linked amelogenesis imperfecta (AIH1)

A hallmark of biological systems is a reliance on protein assemblies to perform complex functions. We have focused attention on mammalian enamel formation because it relies on a self-assembling protein complex to direct mineral habit. The principle protein of enamel is amelogenin, a 180-amino acid hydrophobic protein that self-assembles to form nanospheres. We have used independent technical methods, consisting of the yeast two-hybrid (Y2H) assay and surface plasmon resonance (SPR), to demonstrate the importance of amelogenin self-assembly domains. In addition, we have analyzed mutations in amelogenin observed in patients with amelogenesis imperfecta who demonstrate defects in enamel formation. Assessments of self-assembly of these mutant amelogenins by either SPR or Y2H assay yield concordant data. These data support the conclusion that the amelogenin amino-terminal self-assembly domain is essential to the creation of an enamel extracellular organic matrix capable of directing mineral formation. It also suggests that a pathway through which point mutations in the amelogenin protein can adversely impact on the formation of the enamel organ is by disturbing self-assembly of the organic matrix. These data support the utilization of the Y2H assay to search for protein interactions among extracellular matrix proteins that contribute to biomineralization and provide functional information on protein-protein and protein-mineral interactions.