The energetic basis for hydroxyapatite mineralization by amelogenin variants provides insights into the origin of amelogenesis imperfecta.

Small variations in the primary amino acid sequence of extracellular matrix proteins can have profound effects on the biomineralization of hard tissues. For example, a change in one amino acid within the amelogenin protein can lead to drastic changes in enamel phenotype, resulting in amelogenesis imperfecta, enamel that is defective and easily damaged. Despite the importance of these undesirable phenotypes, there is very little understanding of how single amino acid variation in amelogenins can lead to malformed enamel. Here, we aim to develop a thermodynamic understanding of how protein variants can affect steps of the biomineralization process. High-resolution, in situ atomic force microscopy (AFM) showed that altering one amino acid within the murine amelogenin sequence (natural variants T21 and P41T, and experimental variant P71T) resulted in an increase in the quantity of protein adsorbed onto hydroxyapatite (HAP) and the formation of multiple protein layers. Quantitative analysis of the equilibrium adsorbate amounts revealed that the protein variants had higher oligomer-oligomer binding energies. MMP20 enzyme degradation and HAP mineralization studies showed that the amino acid variants slowed the degradation of amelogenin by MMP20 and inhibited the growth and phase transformation of HAP. We propose that the protein variants cause malformed enamel because they bind excessively to HAP and disrupt the normal HAP growth and enzymatic degradation processes. The in situ methods applied to determine the energetics of molecular level processes are powerful tools toward understanding the mechanisms of biomineralization.

Solid-State NMR Identification of Intermolecular Interactions in Amelogenin Bound to Hydroxyapatite.

Biomineralization processes govern the formation of hierarchical hard tissues such as bone and teeth in living organisms, and mimicking these processes could lead to the design of new materials with specialized properties. However, such advances require structural characterization of the proteins guiding biomineral formation to understand and mimic their impact. In their "active" form, biomineralization proteins are bound to a solid surface, severely limiting our ability to use many conventional structure characterization techniques. Here, solid-state NMR spectroscopy was applied to study the intermolecular interactions of amelogenin, the most abundant protein present during the early stages of enamel formation, in self-assembled oligomers bound to hydroxyapatite. Intermolecular dipolar couplings were identified that support amelogenin dimer formation stabilized by residues toward the C-termini. These dipolar interactions were corroborated by molecular dynamics simulations. A ß-sheet structure was identified in multiple regions of the protein, which is otherwise intrinsically disordered in the absence of hydroxyapatite. To our knowledge, this is the first intermolecular protein-protein interaction reported for a biomineralization protein, representing an advancement in understanding enamel development and a new general strategy toward investigating biomineralization proteins.

The leucine-rich amelogenin protein (LRAP) is primarily monomeric and unstructured in physiological solution.

Amelogenin proteins are critical to the formation of enamel in teeth and may have roles in controlling growth and regulating microstructures of the intricately woven hydroxyapatite (HAP). Leucine-rich amelogenin protein (LRAP) is a 59-residue splice variant of amelogenin and contains the N- and C-terminal charged regions of the full-length protein thought to control crystal growth. Although the quaternary structure of full-length amelogenin in solution has been well studied and can consist of self-assemblies of monomers called nanospheres, there is limited information on the quaternary structure of LRAP. Here, sedimentation velocity analytical ultracentrifugation (SV) and small angle neutron scattering (SANS) were used to study the tertiary and quaternary structure of LRAP at various pH values, ionic strengths, and concentrations. We found that the monomer is the dominant species of phosphorylated LRAP (LRAP(+P)) over a range of solution conditions (pH 2.7-4.1, pH 4.5-8, 50 mmol/L(mM) to 200 mM NaCl, 0.065-2 mg/mL). The monomer is also the dominant species for unphosphorylated LRAP (LRAP(-P)) at pH 7.4 and for LRAP(+P) in the presence of 2.5 mM calcium at pH 7.4. LRAP aggregates in a narrow pH range near the isoelectric point of pH 4.1. SV and SANS show that the LRAP monomer has a radius of ∼2.0 nm and an asymmetric structure, and solution NMR studies indicate that the monomer is largely unstructured. This work provides new insights into the secondary, tertiary, and quaternary structure of LRAP in solution and provides evidence that the monomeric species may be an important functional form of some amelogenins.

Beyond the active site: the impact of the outer coordination sphere on electrocatalysts for hydrogen production and oxidation.

Redox active metalloenzymes play a major role in energy transformation reactions in biological systems. Examples include formate dehydrogenases, nitrogenases, CO dehydrogenase, and hydrogenases. Many of these reactions are also of interest to humans as potential energy storage or utilization reactions for photoelectrochemical, electrolytic, and fuel cell applications. These metalloenzymes consist of redox active metal centers where substrates are activated and undergo transformation to products accompanied by electron and proton transfer to or from the substrate. These active sites are typically buried deep within a protein matrix of the enzyme with channels for proton transport, electron transport, and substrate/product transport between the active site and the surface of the protein. In addition, there are amino acid residues that lie in close proximity to the active site that are thought to play important roles in regulating and enhancing enzyme activity. Directly studying the outer coordination sphere of enzymes can be challenging due to their complexity, and the use of modified molecular catalysts may allow us to provide some insight. There are two fundamentally different approaches to understand these important interactions. The "bottom-up" approach involves building an amino acid or peptide containing outer coordination sphere around a functional molecular catalyst, and the "top-down" approach involves attaching molecular catalyst to a structured protein. Both of these approaches have been undertaken for hydrogenase mimics and are the emphasis of this Account. Our focus has been to utilize amino acid or peptide based scaffolds on an active functional enzyme mimic for H2 oxidation and production, [Ni(P(R)2N(R('))2)2](2+). This "bottom-up" approach has allowed us to evaluate individual functional group and structural contributions to electrocatalysts for H2 oxidation and production. For instance, using amine, ether, and carboxylic acid functionalities in the outer coordination sphere enhances proton movement and results in lower catalytic overpotentials for H2 oxidation, while achieving water solubility in some cases. Amino acids with acidic and basic side chains concentrate substrate around catalysts for H2 production, resulting in up to 5-fold enhancements in rate. The addition of a structured peptide in an H2 production catalyst limited the structural freedom of the amino acids nearest the active site, while enhancing the overall rate. Enhanced stability to oxygen or extreme conditions such as strongly acidic or basic conditions has also resulted from an amino acid based outer coordination sphere. From the "top-down" approach, others have achieved water solubility and photocatalytic activity by associating this core complex with photosystem-I. Collectively, by use of this well understood core, the role of individual and combined features of the outer coordination sphere are starting to be understood at a mechanistic level. Common mechanisms have yet to be defined to predictably control these processes, but our growing knowledge in this area is essential for the eventual mimicry of enzymes by efficient molecular catalysts for practical use.

Improved protocol to purify untagged amelogenin - Application to murine amelogenin containing the equivalent P70→T point mutation observed in human amelogenesis imperfecta.

Amelogenin is the predominant extracellular protein responsible for converting carbonated hydroxyapatite into dental enamel, the hardest and most heavily mineralized tissue in vertebrates. Despite much effort, the precise mechanism by which amelogenin regulates enamel formation is not fully understood. To assist efforts aimed at understanding the biochemical mechanism of enamel formation, more facile protocols to purify recombinantly expressed amelogenin, ideally without any tag to assist affinity purification, are advantageous. Here we describe an improved method to purify milligram quantities of amelogenin that exploits its high solubility in 2% glacial acetic acid under conditions of low ionic strength. The method involves heating the frozen cell pellet for two 15min periods at ∼70°C with 2min of sonication in between, dialysis twice in 2% acetic acid (1:250 v/v), and reverse phase chromatography. A further improvement in yield is obtained by resuspending the frozen cell pellet in 6M guanidine hydrochloride in the first step. The acetic acid heating method is illustrated with a murine amelogenin containing the corresponding P70→T point mutation observed in an human amelogenin associated with amelogenesis imperfecta (P71T), while the guanidine hydrochloride heating method is illustrated with wild type murine amelogenin (M180). The self-assembly properties of P71T were probed by NMR chemical shift perturbation studies as a function of protein (0.1-1.8mM) and NaCl (0-367mM) concentration. Relative to similar studies with wild type murine amelogenin, P71T self-associates at lower protein or salt concentrations with the interactions initiated near the N-terminus.

A solution NMR investigation into the impaired self-assembly properties of two murine amelogenins containing the point mutations T21→I or P41→T.

Amelogenesis imperfecta describes a group of inherited disorders that results in defective tooth enamel. Two disorders associated with human amelogenesis imperfecta are the point mutations T21→I or P40→T in amelogenin, the dominant protein present during the early stages of enamel biomineralization. The biophysical properties of wildtype murine amelogenin (M180) and two proteins containing the equivalent mutations in murine amelogenin, T21→I (M180-I) and P41→T (M180-T), were probed by NMR spectroscopy. At low protein concentration (0.1mM), M180, M180-I, and M180-T are predominately monomeric at pH 3.0 in 2% acetic acid and neither mutation produces a major structural change. Chemical shift perturbation studies as a function of protein (0.1-1.8mM) or NaCl (0-400mM) concentrations show that the mutations affect the self-association properties by causing self-assembly at lower protein or salt concentrations, relative to wildtype amelogenin, with the largest effect observed for M180-I. Under both conditions, the premature self-assembly is initiated near the N-terminus, providing further evidence for the importance of this region in the self-assembly process. The self-association of M180-I and M180-T at lower protein concentrations and lower ionic strengths than wildtype M180 may account for the clinical phenotypes of these mutations, defective enamel formation.

The structure, dynamics, and energetics of protein adsorption-lessons learned from adsorption of statherin to hydroxyapatite.

Proteins are found to be involved in interaction with solid surfaces in numerous natural events. Acidic proteins that adsorb to crystal faces of a biomineral to control the growth and morphology of hard tissue are only one example. Deducing the mechanisms of surface recognition exercised by proteins has implications to osteogenesis, pathological calcification and other proteins functions at their adsorbed state. Statherin is an enamel pellicle protein that inhibits hydroxyapatite nucleation and growth, lubricates the enamel surface, and is recognized by oral bacteria in periodontal diseases. Here, we highlight some of the insights we obtained recently using both thermodynamic and solid state NMR measurements to the adsorption process of statherin to hydroxyapatite. We combine macroscopic energy characterization with microscopic structural findings to present our views of protein adsorption mechanisms and the structural changes accompanying it and discuss the implications of these studies to understanding the functions of the protein adsorbed to the enamel surfaces.

The nucleation and growth of calcium phosphate by amelogenin.

The nucleation processes involved in calcium phosphate formation in tooth enamel are not well understood but are believed to involve proteins in the extracellular matrix. The ability of one enamel protein, amelogenin, to promote the nucleation and growth of calcium phosphate was studied in an in vitro system involving metastable supersaturated solutions. It was found that recombinant amelogenin (rM179 and rp(H)M180) promoted the nucleation of calcium phosphate compared to solutions without protein. The amount of calcium phosphate increased with increasing supersaturation of the solutions and increasing protein concentrations up to 6.5 µg/mL. At higher protein concentrations, the amount of calcium phosphate decreased. The kinetics of nucleation was studied in situ and in real time using a quartz crystal microbalance (QCM) and showed that the protein reduced the induction time for nucleation compared to solutions without protein. This work shows a nucleation role for amelogenin in vitro which may be promoted by the association of amelogenin into nanosphere templates, exposing charged functionality at the surface.

1H, 13C, and 15N resonance assignments of murine amelogenin, an enamel biomineralization protein.

Amelogenin is the predominant matrix protein in developing dental enamel. Making extensive use of residue specific 15N-labeled amino acids samples, the majority of the main and side chain resonances for murine amelogenin were assigned in 2% aqueous acetic acid at pH 3.0.

The COOH terminus of the amelogenin, LRAP, is oriented next to the hydroxyapatite surface.

The organic matrix in forming enamel consists largely of the amelogenin protein self-assembled into nanospheres that are necessary to guide the formation of the unusually long and highly ordered hydroxyapatite (HAP) crystallites that constitute enamel. Despite its ability to direct crystal growth, the interaction of the amelogenin protein with HAP is unknown. However, the demonstration of growth restricted to the c-axis suggests a specific protein-crystal interaction, and the charged COOH terminus is often implicated in this function. To elucidate whether the COOH terminus is important in the binding and orientation of amelogenin onto HAP, we have used solid state NMR to determine the orientation of the COOH terminus of an amelogenin splice variant, LRAP (leucine-rich amelogenin protein), which contains the charged COOH terminus of the full protein, on the HAP surface. These experiments demonstrate that the methyl 13C-labeled side chain of Ala46 is 8.0 A from the HAP surface under hydrated conditions, for the protein with and without phosphorylation. The experimental results provide direct evidence orienting the charged COOH-terminal region of the amelogenin protein on the HAP surface, optimized to exert control on developing enamel crystals.

Molecular recognition at the protein-hydroxyapatite interface.

Proteins found in mineralized tissues act as nature's crystal engineers, where they play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite (bones/teeth) and calcium oxalate (kidney stones). Despite their importance in hard-tissue formation and remodeling, and in pathological processes such as stone formation and arterial calcification, there is little known of the protein structure-function relationships that govern hard-tissue engineering. Here we review early studies that have utilized solid-state NMR (ssNMR) techniques to provide in situ secondary-structure determination of statherin and statherin peptides on their biologically relevant hydroxyapatite (HAP) surfaces. In addition to direct structural study, molecular dynamics studies have provided considerable insight into the protein-binding footprint on hydroxyapatite. The molecular insight provided by these studies has also led to the design of biomimetic fusion peptides that utilize nature's crystal-recognition mechanism to display accessible and dynamic bioactive sequences from the HAP surface. These peptides selectively engage adhesion receptors and direct specific outside-in signaling pathway activation in osteoblast-like cells.