Weilong Zhao

A potential mechanism for amino acid-controlled crystal growth of hydroxyapatite

The mineral component of bone, dentin and calcified parts of avian tendon, hydroxyapatite (HAP), has non-stoichiometric composition (idealized as Ca10(PO4)6(OH)2), plate-like morphology and nanometer size. This unique crystal morphology contributes to the physico-chemical and biochemical properties of bone. Thus, understanding the mechanism for the controlled growth of plate-like HAP nanocrystals is significant in the study of bone biomineralization. Previous studies have shown that acidic non-collagenous proteins (ANCPs), which are enriched in the residues of acidic amino acids, may play an important role in HAP crystal growth modulation. In this study, glutamic acid (Glu) and phosphoserine (Ser-OPO3) were used as model compounds to modify the synthesis of HAP nanocrystals. To identify the mechanisms of amino acids as regulators, X-ray diffraction (XRD), transmission electron microscopy (TEM) and solid state nuclear magnetic resonance (ssNMR) were used. The crystals obtained in the inorganic controls were needle-like, while crystals synthesized in the presence of the amino acids presented a plate-like morphology. The plate-like crystals had a preferred crystal orientation on (300) face, which was lacking in the inorganically grown crystals, indicating preferential adsorption and suppression of growth in specific crystal directions. Ser-OPO3 was more efficient than Glu in modulating HAP nucleation and crystal growth. Furthermore, NMR revealed interactions between the charged side chain groups in amino acids and the crystal surfaces. These results were successfully explained through our MD simulations for the free energy calculation of amino acid binding on HAP crystal faces. The present study revealed that amino acids may act as effective regulators of HAP morphology without the need to invoke large NCPs in bone biomineralization and in designing bioinspired materials for orthopaedic and dental applications.

Predicting the Structure–Activity Relationship of Hydroxyapatite-Binding Peptides by Enhanced-Sampling Molecular Simulation

Understanding the molecular structural and energetic basis of the interactions between peptides and inorganic surfaces is critical to their applications in tissue engineering and biomimetic material synthesis. Despite recent experimental progresses in the identification and functionalization of hydroxyapatite (HAP)-binding peptides, the molecular mechanisms of their interactions with HAP surfaces are yet to be explored. In particular, the traditional method of molecular dynamics (MD) simulation suffers from insufficient sampling at the peptide-inorganic interface that renders the molecular-level observation dubious. Here we demonstrate that an integrated approach combining bioinformatics, MD, and metadynamics provides a powerful tool for investigating the structure-activity relationship of HAP-binding peptides. Four low charge density peptides, previously identified by phage display, have been considered. As revealed by bioinformatics and MD, the binding conformation of the peptides is controlled by both the sequence and the amino acid composition. It was found that formation of hydrogen bonds between lysine residue and phosphate ions on the surface dictates the binding of positively charged peptide to HAP. The binding affinities of the peptides to the surface are estimated by free energy calculation using parallel-tempering metadynamics, and the results compare favorably to measurements reported in previous experimental studies. The calculation suggests that the charge density of the peptide primarily controls the binding affinity to the surface, while the backbone secondary structure that may restrain side chain orientation toward the surface plays a minor role. We also report that the application of enhanced-sampling metadynamics effects a major advantage over the steered MD method by significantly improving the reliability of binding free energy calculation. In general, our novel integration of diverse sampling techniques should contribute to the rational design of surface-recognition peptides in biomedical applications.

Isoexergonic Conformations of Surface-Bound Citrate Regulated Bioinspired Apatite Nanocrystal Growth

The superior biomechanical properties of bone and dentin are dictated, in part, by the unique plate-like morphology of hydroxyapatite (HAP) nanocrysals within a hierarchically assembled collagen matrix. Understanding the mechanism of crystal growth and thus morphology is important to the rational design of bioinspired apatite nanocrystals for orthopedic and dental applications. Citrate has long been proposed to modulate apatite crystal growth, but major questions exist regarding the HAP-bound citrate conformations and the identities of the interacting functional groups and HAP surface sites. Here, we conducted a comprehensive investigation of the mechanism from the angstrom to submicrometer scale by detailed correlation of the results of high-level metadynamics simulations, employing force-fields benchmarked to experiment and density functional theory calculations with the results of high resolution transmission electron microscopy, nuclear magnetic resonance spectroscopy, solution analysis, and thermogravimetric analysis. Crystal morphology changed from needle- to plate-like with increasing citrate concentration. Citrate adsorbed more strongly on the HAP (100) face than on the (001) face, thus resulting in preferential growth in the [001] direction and the plate-like morphology. Two very different bound conformations were obtained, involving interactions of either one or both terminal carboxyl groups with three or five surface calcium ions, respectively, and a hydrogen bond between the citrate hydroxyl and the HAP surface. Remarkably, despite fewer interaction sites in the single bound carboxyl conformation, the structures were isoexergonic, so both exist at equilibrium. Identification of the former conformation is significant because it allows a greater adsorption density than is traditionally assumed and can help explain concentration-dependence of citrate in modulating crystal morphology. These unique results were enabled first by the application of advanced metadynamics, a technique necessary for the accurate simulation of ionic materials but which is rarely employed in the biomaterials and biomineralization fields and second by the detailed correlation of computational, spectroscopic, and analytical results.

Method Evaluations for Adsorption Free Energy Calculations at the Solid/Water Interface through Metadynamics, Umbrella Sampling, and Jarzynski's Equality

Considerable interest in characterizing protein/peptide-surface interactions has prompted extensive computational studies on calculations of adsorption free energy. However, in many cases, each individual study has focused on the application of free energy calculations to a specific system; therefore, it is difficult to combine the results into a general picture for choosing an appropriate strategy for the system of interest. Herein, three well-established computational algorithms are systemically compared and evaluated to compute the adsorption free energy of small molecules on two representative surfaces. The results clearly demonstrate that the characteristics of studied interfacial systems have crucial effects on the accuracy and efficiency of the adsorption free energy calculations. For the hydrophobic surface, steered molecular dynamics exhibits the highest efficiency, which appears to be a favorable method of choice for enhanced sampling simulations. However, for the charged surface, only the umbrella sampling method has the ability to accurately explore the adsorption free energy surface. The affinity of the water layer to the surface significantly affects the performance of free energy calculation methods, especially at the region close to the surface. Therefore, a general principle of how to discriminate between methodological and sampling issues based on the interfacial characteristics of the system under investigation is proposed.

Quantitatively Identifying the Roles of Interfacial Water and Solid Surface in Governing Peptide Adsorption

Understanding the molecular mechanism of protein adsorption on solids is critical to their applications in materials synthesis and tissue engineering. Although the water phase at the surface/water interface has been recognized as three types: bulk water, intermediate water phase and surface-bound water layers, the roles of the water and surface in determining the protein adsorption are not clearly identified, particularly at the quantitative level. Herein, we provide a methodology involving the combination of microsecond strengthen sampling simulation and force integration to quantitatively characterize the water-induced contribution and the peptide-surface interactions into the adsorption free energy. Using hydroxyapatite and graphene surfaces as examples, we demonstrate how the distinct interfacial features dominate the delicate force balance between these two thermodynamics parameters, leading to surface preference/resistance to peptide adsorption. Specifically, the water layer provides sustained repelling force against peptide adsorption, as indicated by a monotonic increase in the water-induced free energy profile, whereas the contribution from the surface-peptide interactions is thermodynamically favorable to peptide adsorptions. More importantly, the revealed adsorption mechanism is critically dictated by the distribution of water phase, which plays a crucial role in establishing the force balance between the interactions of the peptide with the water layer and the surface. For the HAP surface, the charged peptide exhibits strong binding affinity to the surface, due to the controlling contribution of peptide-surface interaction in the intermediate water phase. The surface-bound water layers are observed as the origin of bioresistance of solid surfaces toward the adsorption of charge-neutral peptides. The preferred peptide adsorption on the graphene, however, is dominated by the surface-induced component at the water layers adjacent to the surface. Our results further elucidate that the intermediate water phase significantly shortens the effective range of the surface dispersion force, in contrast to the observation on the hydrophilic surface.