Adsorption Processes of Gly and Glu Amino Acids on Hydroxyapatite

Jul 21, 2007 - Zhijun XuXiao YangQichao WeiWeilong ZhaoBeiliang CuiXiaoning YangNita Sahai .... Langmuir 0 (proofing), ... Wenge Jiang , Haihua Pan , ...
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Langmuir 2007, 23, 8972-8981

Adsorption Processes of Gly and Glu Amino Acids on Hydroxyapatite Surfaces at the Atomic Level Haihua Pan, Jinhui Tao, Xurong Xu, and Ruikang Tang* Department of Chemistry and Center for Biomaterials and Biopathways, Zhejiang UniVersity, Hangzhou, 31027, China ReceiVed February 26, 2007. In Final Form: April 17, 2007 The regulation mechanism of organic additives on the crystallization of inorganic crystal is fundamentally important in biomineralization. Experimentally, it was found that the amino acids glycine (Gly) and glutamic acid (Glu) could lead to the formation of rod- and plate-like hydroxyapatite (HAP) crystallites, respectively. The detailed adsorption behavior of Gly and Glu on HAP crystal faces was studied by molecular dynamics (MD) simulation. The specific adsorption sites and patterns of Gly and Glu on the (100) and (001) faces of HAP crystals were revealed at the atomic level. The amino acids adsorbed on the HAP (001) and (100) faces with their positive amino groups occupied vacant calcium sites, and their negative carboxylate groups occupied vacant P or OH sites precisely and formed an ordered adsorption layer. The atomic force microscopy pulling simulation and free energy calculation showed that Glu was much more difficult to depart from the HAP (001) face than that from the (100) face. This result indicated that Glu preferred to adsorb strongly onto the HAP (001) face, which resulted in the formation of plate-like HAP. However, Gly did not show any significantly preferential adsorption between these two HAP faces. Thus, the habits of HAP, rod-like crystallites, were not altered during the HAP crystallization in the presence of Gly. Combined with experimental results, our study demonstrated that the MD simulation of interfacial structures could improve our understanding of biological regulation in mineralization processes at the atomic level.

1. Introduction The organic-inorganic interface is of importance since it provides the active sites for biological control in biomineralizations. An important but largely unresolved issue is the way in which nature controls the nucleation, growth, and morphology of inorganic crystallites and the function of biomolecules (such as amino acids and proteins) in these reactions. A generally accepted mechanism of crystallization is that the ions precipitate on the crystal surface through diffusion, adatom, and/or surface diffusion to the kink sites,1,2 which is called an ion-by-ion attachment mechanism. It is suggested that the biomolecules may adsorb on or in the vicinity of the crystal surface to inhibit or promote the ion attachments on certain crystal faces. Thus, the kinetics of crystallization and the resulting habits of crystals can be regulated. Hydroxyapatite (HAP, Ca10(PO4)6(OH)2) is considered to be an important model for biominerals3 since it is the major inorganic component of bones and teeth.4 Many bone or dental related proteins contain large amounts of acidic residues, which are believed to play important roles in the modification of mineral precipitation.5-12 As compared with complicated proteins, amino * Corresponding author. E-mail: [email protected]; tel./fax: + 86-57187953736. (1) Burton, W. K.; Cabrera, N.; Frank, F. C. Philos. Trans. R. Soc. London, Ser. A 1951, 243, 299. (2) Lasaga, A. C. Theory in the Earth Sciences; Princeton University Press: Princeton, NJ, 1998. (3) Mathew, M.; Takagi, S. J. Res. Natl. Inst. Stand. Technol. 2001, 106, 1035. (4) Mann, S. Biomineralization; Principles and Concepts in Bioinorganic Materials Chemistry; Oxford University Press: New York, 2001. (5) Chen, C.; Boskey, A. L.; Rosenberg, L. C. Calcif. Tissue Int. 1984, 36, 285. (6) Dalas, E.; Ioannou, P. V.; Koutsoukos, P. G. Langmuir 1989, 5, 157. (7) Dalas, E.; Ioannou, P. V.; Koutsoukos, P. G. Langmuir 1990, 6, 535. (8) Fujisawa, R.; Kuboki, Y.; Nancollas, G. H. Biochim. Biophys. Acta 1991, 1075, 56. (9) Long, J. R.; Dindot, J. L.; Zebroski, H.; Kiihne, S. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12083. (10) Raj, P. A.; Johnsson, M.; Levine, M. J.; Nancollas, G. H. J. Biol. Chem. 1992, 267, 5968.

acids are also reported to be effective regulators in HAP formation.13-17 X-ray absorption near edge structure (XANES) spectroscopy12 and solid-state NMR (ssNMR)11,18 can provide evidence for possible biomolecular interaction sites at the organic-inorganic interface. However, detailed structural information such as adsorption and interaction sites and patterns of biomolecules on the HAP crystal faces is poorly understood. Molecular dynamics (MD) simulations are a useful tool for modeling and probing the structure and adsorption of organic molecules on inorganic crystals at the atomic level.19-25 Some useful information, such as interfacial energy,26 adsorption energy,19 and interaction sites,21 also can be depicted by MD simulations to establish organic-inorganic interfaces. Recently, a developed ab initio method has been applied successfully to simulate the adsorption of Gly at the pyrite-water interface;22 however, tremendous computing resources are required for the large system (>8000 atoms) and the relatively long simulation time (>2 ns) by the ab initio method. Previously, empirical force fields have been established for the simulation of apatite crystals.23,25,27-29 The simulation results can be in good agreement with the real crystal structure,23,27-30 IR spectra,27 and molar (11) Shaw, W. J.; Campbell, A. A.; Paine, M. L.; Snead, M. L. J. Biol. Chem. 2004, 279, 40263. (12) Gilbert, P. U. P. A.; Abrecht, M.; Frazer, B. H. ReV. Miner. Geochem. 2005, 59, 157. (13) Matsumoto, T.; Okazaki, M.; Inoue, M.; Hamada, Y.; Taira, M.; Takahashi, J. Biomaterials 2002, 23, 2241. (14) Koutsopoulos, S.; Dalas, E. Langmuir 2000, 16, 6739. (15) Koutsopoulos, S.; Dalas, E. Langmuir 2001, 17, 1074. (16) Koutsopoulos, S.; Dalas, E. J. Cryst. Growth 2000, 217, 410. (17) Koutsopoulos, S.; Dalas, E. J. Colloid Interface Sci. 2000, 231, 207. (18) Stayton, P. S.; Drobny, G. P.; Shaw, W. J.; Long, J. R.; Gilbert, M. Crit. ReV. Oral. Biol. Med. 2003, 14, 370. (19) Harding, J.; Duffy, D. J. Mater. Chem. 2006, 16, 1105. (20) Cooper, T. G.; de Leeuw, N. H. Langmuir 2004, 20, 3984. (21) de Leeuw, N. H.; Cooper, T. G. Cryst. Growth Des. 2004, 4, 123. (22) Nair, N. N.; Schreiner, E.; Marx, D. J. Am. Chem. Soc. 2006, 128, 13815. (23) de Leeuw, N. Phys. Chem. Chem. Phys. 2004, 6, 1860. (24) de Leeuw, N.; Mkhonto, D. Chem. Mater. 2003, 15, 1567. (25) Mkhonto, D.; de Leeuw, N. J. Mater. Chem. 2002, 12, 2633. (26) Duffy, D. M.; Harding, J. H. Langmuir 2004, 20, 7637.

10.1021/la700567r CCC: $37.00 © 2007 American Chemical Society Published on Web 07/21/2007

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enthalpy, etc.29,31,32 The interfacial structures of the apatitewater interface33,34 also have been studied by using this method. MD simulations may provide another view of how amino acids adsorb and interact on/with the HAP faces at the atomic level, which would explain their effect on crystal habits. The departure forces and difference of free energy of the amino acids from their adsorption sites can also be examined by AFM (atomic force microscopy) pulling (or steered MD35) simulations, which reflect the adsorption affinities of these molecules onto the individual HAP faces. Thus, the detailed regulation mechanism of HAP crystallization with the help of additives can be probed by MD simulations. 2. Computational Details 2.1. Interfacial Structures and Models. Typical biological apatite has a hexagonal crystal structure (space group P63/m).36,37 From crystal structure determinations of hexagonal HAP, an occupation of 0.5 for each crystallographic OH- position was obtained as an average over the whole crystal.36 The orientation of these hydroxide ions within the channels formed by calcium triangles (these calcium ions are called Ca2, or triangular calcium, and the other calcium ions lie in a column, which are called as Ca1, or column calcium) was treated as follows: half of the hydroxide ions were up or half were down randomly in the c direction for all calcium triangle channels. For clarity, the surface position of hydroxide ions, Ca1 ions, Ca2 ions, and phosphate ions according to the crystal structure were labeled as OH, Ca1, Ca2, and P sites, respectively. A block of 4 × 4 × 5 unit cells (37.7 Å × 37.7 Å × 34.4 Å) of hexagonal HAP was built as the simulation model. The initial configuration was taken from the X-ray crystal structure (a ) b ) 9.423 Å, c ) 6.883 Å, R ) β ) 90°, g ) 120°).36 Usually, the individual crystal faces were obtained by cutting the crystal along the required crystallographic direction. The common rule for the cutting was that the formed planes were electrically neutral or the dipole moment was removed from polar directions by adding/removing ions (or making some defects on the cutting planes).26 Since HAP is a polar crystal, the HAP (001) surfaces were created by splitting the crystal structure at the middle of the Ca2+rich (Ca-rich) layers (Figure 1A, c type) in this work, each plane contained half of the Ca2+ ions, which was also applied by Zahn and Hochrein.33 According to the HRTEM results of the HAP (100) boundary of crystal amorphous38 and X-ray reflection results of apatite in solution,39 the cutting plane for the HAP (100) surfaces was selected over the OH- channel and by adding Ca2+ ions on the PO43- columns to remove the excess surface charge (Figure 1B, e type). A slab of 3 nm (large enough to avoid size effects) water was introduced between the separated crystal surfaces with a density of 1000 kg/m3, and the HAP (001) or (100) interfaces in water were thus established. Koutsopoulos and Dalas14-17 reported that a Langmuir type of adsorption behavior was found for amino acids in the kinetic studies of HAP crystal growth. For simplicity, different (27) Hauptmann, S.; Dufner, H.; Brickmann, J.; Kast, S.; Berry, R. Phys. Chem. Chem. Phys. 2003, 5, 635. (28) Meis, C.; Gale, J. D.; Boyer, L.; Carpena, J.; Gosset, D. J. Phys. Chem. A 2000, 104, 5380. (29) Cruz, F. J. A. L.; Lopes, J. N. C.; Calado, J. C. G.; da Piedade, M. E. M. J. Phys. Chem. B 2005, 109, 24473. (30) Mkhonto, D.; de Leeuw, N. J. Mater. Chem. 2002, 12, 2633. (31) Cruz, F. J. A. L.; Lopes, J. N. C.; Calado, J. C. G. Fluid Phase Equilib. 2006, 241, 51. (32) Cruz, F. J. A. L.; Lopes, J. N. C.; Calado, J. C. G. J. Phys. Chem. B 2006, 110, 4387. (33) Zahn, D.; Hochrein, O. Phys. Chem. Chem. Phys. 2003, 5, 4004. (34) Pan, H.; Tao, J.; Wu, T.; Tang, R. Chin. J. Inorg. Chem. 2006, 22, 1392. (35) Isralewitz, B.; Gao, M.; Schulten, K. Curr. Opin. Struct. Biol. 2001, 11, 224. (36) Wilson, R.; Elliott, J.; Dowker, S. Am. Mineral. 1999, 84, 1406. (37) Narasaraju, T. S. B.; Phebe, D. E. J. Mater. Sci. 1996, 31, 1. (38) Sato, K.; Kogure, T.; Iwai, H.; Tanaka, J. J. Am. Ceram. Soc. 2002, 85, 3054. (39) Park, C.; Fenter, P.; Zhang, Z.; Cheng, L.; Sturchio, N. Am. Mineral. 2004, 89, 1647.

Figure 1. Cutting planes of HAP (001) and (100) faces. (A) Cutting planes are between calcium- and phosphate-rich layers ((001) a/b type) or along the calcium-rich layers ((001) c/c type). (B) Cutting planes are close to the hydroxyl column ((100) a/b type) or between the phosphate-rich layers ((100) c/c type) or Ca1 ion redistributed type ((100) d/e type). amounts (4-64) of amino acids were added to the HAP-water interfaces (the total surface area was about 24-25 Å2 in the simulations) to make a system with different surface coverages of amino acids. Sodium ions were also added to make the whole system of the HAP solution neutral. 2.2. Simulation Methods. In our simulation, the semi-flexible apatite model of Hauptmann et al.,27 which reproduced the experimental crystal parameters and infrared spectra very well, was used, in which the intermolecular interactions were described by a combination of Coulomb and Born-Mayer-Huggins potential (BMH, as described in eq 1) terms. For HAP-water systems and HAP-water-amino acid systems, the simple SPC40 force fields, widely used in the modeling of large biological aqueous systems, were applied for water; OPLS-AA41 force fields, commonly used in biological systems, were applied for amino acids. For both SPC and OPLS-AA force fields, the dispersion term was described by the Lennard-Jones potential (LJ, described in eq 2). Since the mixing rules were not given for BMH and LJ potential terms, the BMH potential term was then transferred into the LJ potential term by fitting these two terms in the minimum and attraction region of the BMH potential term. Both Hauptmann’s model and the fitted LJ forms of the model reproduced the experimental hexagonal crystal parameters well.34 The force field parameters and simulation results of the HAP crystal parameters are given in the Supporting Information.

] [( ) ( ) ]

Uij ) w(Fi + Fj) exp

Uij ) 4ij

[

CiCj Ri + Rj - rij - 6 Fi + F j rij

σij rij

12

-

σij rij

(1)

6

(2)

The MD simulations were performed using the Gromacs 3.3 package.42,43 Periodic boundary conditions were applied in all directions. PME44,45 summation was applied for treatment of the long-range Coulombic interactions. The cutoff distance was chosen to be 1.3 nm. Our simulations were done in the NpT ensemble at atmospheric pressure and at a temperature of 310 K. The anisotropic (40) Berendsen, H.; Postma, J. P. M.; van Gunsteren, W. F.; Hermans, J. Interaction Models for Water in Relation to Protein Hydration. In Intermolecular Forces; D. Reidel Publishing Company: Dordrecht, The Netherlands, 1981; pp 331-342. (41) Jorgensen, W. L.; Tirado-Rives, J. J. Am. Chem. Soc. 1988, 110, 1657. (42) Berendsen, H.; van der Spoel, D.; van Drunen, R. Comp. Phys. Commun. 1995, 91, 43. (43) Lindahl, E.; Hess, B.; van der Spoel, D. J. Mol. Model. 2001, 7, 306. (44) Darden, T.; York, D.; Pedersen, L. J. Chem. Phys. 1993, 98, 10089. (45) Essmann, U.; Perera, L.; Berkowitz, M.; Darden, T.; Lee, H.; Pedersen, L. J. Chem. Phys. 1995, 103, 8577.

8974 Langmuir, Vol. 23, No. 17, 2007 Berendson pressure coupling46 was applied, allowing cell shape variations so as not to impose artificial symmetry restrictions. A time step of 2 fs was found to be appropriate. Initial velocities according to Maxwell distribution corresponding to the desired temperature were used as the starting configuration. To speed up the adsorption process, a relatively high temperature (810 K) was used in the NVT ensemble before the final NpT ensemble run. About a 3-4 ns NVT ensemble run was found to be appropriate for the amino acids to adsorb on the HAP interfaces in our simulation. After that, a relaxation period of a 500 ps NpT ensemble run was found to be appropriate to ensure convergence of the total energy. To ensure thermodynamic equilibrium, the convergence of the total energy, temperature, pressure, and structures was carefully monitored during the equilibration period. The equilibrium dynamic trajectory for each system was finally recorded for statistical analysis at 100 fs intervals during the next 1 ns of MD simulations. 2.3. AFM Pulling Method. According to the ion-by-ion attachment mechanism, when the crystal surface sites were occupied by additives, the crystal growth on these sites is blocked. The stronger the additive adsorbed, the more effective the inhibitor was. Usually, adhesion energy19 or segregation energy47,48 were used to characterize the absorbability and to explain the morphology of the crystal under the control of templates or additives. But, these methods had some problems with the calculations of the system of asymmetry cut crystal faces (e.g., the HAP (001) d/e faces), in that only mixed (averaged) information of the two different crystal faces was given. The differences of the potential energies of the system with and without additives were hard to calculate precisely because of the large thermal fluctuation. Furthermore, these methods did not take into account the entropy effect, which also might be significant in contributing to the mineral-water interfacial free energy.49 Steered MD simulations35 (or the AFM pulling method, described in Gromacs50) can be used to probe the forces needed to remove the adsorbed amino acids from the HAP crystal faces directly. This is a widely used method in force probes, such as the unbinding of proteins51 and pulling peptides out of membranes.52 In this method, a spring was applied to pull each individual adsorbed amino acid away from the HAP surfaces, and the force was recorded as a function of distance between the ion/molecule and HAP crystal face. The larger departure force implied that it was more difficult for the amino acid to be detached from the surface. The absorbability could also be characterized by the departure force. The reversible work needed to pull the ions out was the difference of free energy.53 From Jarzynski’s equality54,55 and its cumulate expansion approximation (eq 3),53,56 the potential of mean force (PMF), free energy profile along the reaction coordinate, could also be calculated by the mean and variance of the irreversible work of the departure pathway35,51,57 Fλ(τ) - Fλ(0) ) β 1 - log〈exp[-βw(τ)]〉 ≈ 〈w(τ)〉 - (〈w(τ)2〉 - 〈w(τ)〉2) (3) β 2 where Fλ is the free energy at the departure pathway λ at simulation (46) Berendsen, H.; Postma, J. P. M.; DiNola, A.; Haak, J. J. Chem. Phys. 1984, 81, 3684. (47) Rohl, A. L. Curr. Opin. Solid State Mater. Sci. 2003, 7, 21. (48) Braybrook, A. L.; Heywood, B. R.; Jackson, R. A.; Pitt, K. J. Cryst. Growth 2002, 243, 336. (49) Kerisit, S.; Cooke, D. J.; Spagnoli, D.; Parker, S. C. J. Mater. Chem. 2005, 15, 1454. (50) van der Spoel, D.; Lindahl, E.; Hess, B.; van Buuren, A.; Apol, E.; Meulenhoff, P.; Tieleman, D.; Sijbers, A. L. T. M.; Feenstra, K. A.; van Drunen, R.; Berendsen, H. Gromacs User Manual, version 3.2.; Groningen, The Netherlands, 2004; http://www.gromacs.org. (51) Lu, H.; Isralewitz, B.; Krammer, A.; Vogel, V.; Schulten, K. Biophys. J. 1998, 75, 662. (52) Contera, S. A.; Lemaitre, V.; de Planque, M. R. R.; Watts, A.; Ryan, J. F. Biophys. J. 2005, 89, 3129. (53) Park, S. Extracting Equlibrium from Nonequilibrium: Free Energy Calculation from Steered Molecular Dynamics Simulations. Ph.D. Thesis, University of Illinois at Urbana-Champaign, 2004. (54) Jarzynski, C. Phys. ReV. Lett. 1997, 78, 2690. (55) Jarzynski, C. Phys. ReV. E 1997, 56, 5018.

Pan et al. time τ; β ) 1/KbT, which is the inverse temperature; and w(τ) is the total work of pulling. Applying external forces in a simulation would lead to a statistical distribution of the results. Several trajectories were needed to check the consistency of the results and to calculate the PMF. Since only a small number of trajectories could be sampled due to the limitations on computer time, it was important to minimize the fluctuation of the external work so that the PMF could be calculated accurately even with a small number of trajectories. Here, four trajectories were simulated by selecting differently adsorbed amino acids to be pulled. According to some references35,51,53,56 and the try-error tests, a spring constant of 5000 kJ/mol nm2 and pulling velocity of 0.0005 nm/ps were used to ensure the sensitivity of force probing, guiding of the departure pathway, and local structure relaxation. 2.4. Analysis. The HAP interfacial structure could be described by local density profiles, which were analyzed by dividing the simulation box into 1000 slices and calculating the density in each slice. The local density profiles could be analyzed in terms of the density of water, amino group, carboxylate group, and amino acids and ions near the interface.

3. Experimental Procedures The precipitation of HAP crystallites in the absence and presence of Gly/Glu was performed at a temperature of 37 ( 0.5 °C. A total of 30 mL of 3.2 mM CaCl2 (analytical grade, A.R.) was premixed with 30 mL of 40.0 mM glycine (A.R.) or L-glutamic acid (A.R.) solutions. A total of 60 mL of 0.96 mM Na2HPO4 (A.R.) solution was dropped into the calcium solution at a rate of 1 mL/min with stirring (500 rpm). The pH of the aliquot was maintained at 9.50 ( 0.10 by using 75 mM NaOH (A.R). After the reaction, the suspension was aged for 30 days. About 0.1 mL of aliquot was extracted from the reaction system and dipped onto the copper grid supported by the carbon film, washed with water to remove the residues, and dried in air. All solutions were prepared using triply distilled water. The solids were examined by transmission electron microscopy (TEM, JEM-200CX; JEOL). The samples were placed on the cleaned glass for X-ray diffraction (XRD, D/MAX-RA, Rigaku).

4. Results and Discussion 4.1. Interfacial Structures of HAP with Amino Acids. Simulation work33 has already indicated that there was layered structured water on the HAP (001) and (100) interfaces, which was also proven by experimental work.58-60 It should be mentioned that the (100) and (010) faces of HAP were identical since the crystal had a P63/m space group. The influences of different amounts of adsorbed Gly (represented by different concentrations) on the local density profiles of water (upper part in Figure 2) and amino acids (lower part in Figure 2) on the HAP (001) face were given. In the absence of Gly, the HAP surfaces were surrounded by a highly structured water layer.33 When Gly was introduced into the simulation system, it was found that all the added biomolecules preferred to gather on the HAP surfaces, indicating a stronger interaction or lower free energy of GlyHAP than H2O-HAP. Since some interfacial water molecules were replaced by Gly, the local density of water was decreased. However, even the (001) HAP crystal face was covered by a Gly layer (with a surface concentration of 2.75/nm2), sufficient water molecules still remained on the HAP surface, and their layered structures were also maintained (Figure 2). This phenomenon (56) Schulten, K. Computational Soft Matter: From Synthetic Polymers to Proteins. In Mechanical Functions of Proteins; Attig, N., Binder, K., Grubmu¨ller, H., Kremer, K., Eds.; John von Neumann Institute for Computing: Ju¨lich, Germany, 2004; Vol. 23, pp 423-424. (57) Hummer, G.; Szabo, A. Biophys. J. 2003, 85, 5. (58) Park, C.; Fenter, P.; Zhang, Z.; Cheng, L. W.; Sturchio, N. C. Am. Mineral. 2004, 89, 1647. (59) Wilson, E.; Awonusi, A.; Morris, M.; Kohn, D.; Tecklenburg, M.; Beck, L. Biophys. J. 2006, 90, 3722. (60) Wilson, E.; Awonusi, A.; Morris, M.; Kohn, D.; Tecklenburg, M.; Beck, L. J. Bone Miner. Res. 2005, 20, 625.

Amino Acids on HAP

Figure 2. Local density profiles of water (top) and Gly (bottom) near the HAP (001) interface at different surface coverages of 0/, 0.33/, 0.92/, and 2.75/nm2, respectively. Peak positions of the water density profile of the control system are marked, and some density profiles are shifted or reverted (for Gly). Left region is bulk water, and right one is the HAP (001) face.

showed that although Gly had a greater binding effect on HAP, it could not affect the original structure of interfacial water. It was interesting to note that the density profile of Gly on HAP was quite similar to that of water. When a detailed analysis by the group based local density profile (Figure 3C) was performed and compared with Figure 2, it was found that the first layer was mainly composed of the amino group (N) and that the second layer was mainly composed of the R-carbonate group (C) and carboxylate group (CA). The amino group was trapped in the first water layer, and the R-carbonate group and carboxylate group were trapped in the second water layer (Figures 2 and 3A,C). The Gly molecule was mainly adsorbed on the Ca1 sites with the negatively charged carboxylate group. Each Ca1 site could adsorb three Gly molecules at most (illustrated by the dashed circle in Figure 3B). But, the adsorption pattern for Gly on the HAP (001) face was not obvious. If we supposed that on average, each surface Ca1 site could adsorb 2.5 Gly (one of the Gly was shared by the two adjacent Ca1 sites), the saturated surface concentration of Gly on the HAP (001) face would be 3.33/nm2. As for Glu, the structure of the layered amino acids was also compatible with the layered water structure. The group based local density profile (Figure 5C) indicated that part of the amino group (N) of Glu was adsorbed directly on the HAP interface (which formed the first layer in Figure 4) and that the remaining amino group stayed in the same level with the second water layer (Figure 4). When the HAP interface was covered by a Glu layer (with a surface concentration of 2.25/nm2), the adsorbed Glu molecules formed a line-by-line pattern (marked by the light-green ribbon in Figure 5B) on the HAP (001) face, and the direction of the line was the [010] direction. It should be emphasized that this surface concentration was approaching the saturated surface concentration. If more Glu molecules were added into the system, it was impossible for all the molecules to adsorb on HAP interface simultaneously in the 3-4 ns simulation period. Thus, the maximum coverage of Glu onto the (001) HAP surfaces was about 2.25/nm2. Different from the previous understanding of biomineralization, it was not the whole crystal surface but only some precise sites that were occupied by the additives.

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When Gly was added to the HAP(100)-water interface, it stayed mainly in the region of the second to the fourth peak of water density on the HAP interface (Figure 6). When the surface coverage of Gly reached 3.20/nm2, the first and second water layers almost disappeared as compared to the control condition because the preadsorbed water on the hydroxyl column, which formed the first and second water layers, was driven out by the adsorbed amino group of Gly (Figure 7A). Part of the amino group adsorbed on the HAP interface of the hydroxyl column and the other amino groups occupied the vacant Ca1 sites in the calcium column as shown in the group based local density profile (the first and second layers of the density profile of the amino group, N) (Figure 7C) and the simulation snapshot (Figure 7A). The adsorbed Gly on the HAP (100) face formed a pattern as indicated by the light-green ribbon ([013] direction) and orange dotted line ([0-12] direction) in Figure 7B. The maximum coverage of Gly onto the (100) HAP surfaces was about 3.2/nm2. When Glu was added to the HAP (100) interface, it mainly stayed at the region of the fourth peak of the water density (Figure 8). The group based density profile (Figure 9C) and simulation snapshot (Figure 9A) indicated that carboxylate (terminal carboxylate, C, and side chain carboxylate, CD) adsorbed mainly on the Ca1 column, while the amino group was on both sides of this carboxylate layer to form a N-C-N layered structure, in which the amino group of the inner layer mainly occupied the vacant Ca1 sites or Ca2 sites (around the hydroxyl column). The Glu molecules adsorbed on the HAP (100) faces with a high surface coverage, 2.40/nm2, which approached the saturated surface concentration. On the HAP (100) face, the adsorption patterns of Glu were also observed as that of Gly, and they are indicated by a light-green ribbon ([012] direction) and dashed orange line ([0-13] direction) (Figure 9B). The adsorption of Glu and Gly on the HAP crystal faces also has been confirmed experimentally.13,61 It was found61 that the adsorption amount of Glu on the HAP crystallites was about 4.3 wt %, in which the specific surface area of the HAP sample was ∼86 m2/g. Thus, the estimated surface coverage of Glu on the HAP surface, 2.1/nm2, was consistent with our MD simulation results, 2.2-2.4/nm2. Structured water layers were found on the apatite interfaces by both experimental58-60 and simulation23,30,33,34 studies. In our simulation, we took all the interfacial structures of the HAP interfaces into consideration, and the Gly and Glu adsorbed directly on the HAP interface, replacing the preadsorbed water and changing the original interfacial structures. For both Gly and Glu, the amino group adsorbed more closely to the HAP (001) and (100) faces. There were specific adsorption sites or adsorption patterns for amino acids on the HAP (001) and (100) faces with its positive amino group occupying the vacant Ca1 or Ca2 sites, and its negative carboxylate groups occupied the vacant P or OH sites precisely. The simulation trajectory did not show any apparent lateral or vertical movement of the adsorbed amino acids on the HAP faces in nanosecond simulation, which indicated that there were relatively deep traps or strong interactions for amino acids on specific sites of the HAP faces and in the layered interfacial structure. This behavior could also be demonstrated in the AFM pulling simulation. As the Langmuir kinetic model of crystal growth, these strongly adsorbed amino acids occupied some specific sites of the HAP crystal faces and could result in the inhibition of the attachment of calcium, phosphate, and hydroxyl ions on the HAP surfaces. Thus, the crystallization (61) Boanini, E.; Fini, M.; Gazzano, M.; Bigi, A. Eur. J. Inorg. Chem. 2006, 2006, 4821.

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Figure 3. Simulation snapshot for adsorbed Gly on the HAP (001) face in water with a surface coverage of 2.75/nm2 and group based local density profile. (A) Final configuration in side view. Amino and carboxylate groups are blue (nitrogen) and red (oxygen), respectively. (B) Typical adsorption sites of Gly on the interface. For clarity, water molecules are not shown. (C) Group based density profiles of amino (N), R-carbonate (CA), and carboxylate (C) groups near the HAP interface. (D) Adsorption sites of Gly on the HAP (001) face (top view). Ca1 can catch three Gly at most as marked by the dashed circles. No obvious adsorption pattern is found.

rates of HAP were retarded by these foreign molecules, which has been reported.14,17 4.2. AFM Pulling Simulation. The typical departure force curves for Gly and Glu on specific HAP (001)/c and (100)/e faces are shown in Figure 10. When the amino acids were pulled away from the HAP faces, the forces were increased and then followed by a rupture. There were several rupture forces owing to the multiple structured interfacial layers found in our simulation, which meant that there were multiple traps for amino acids to detach from the HAP faces. These typical zigzag force curves were also found in atomic force microscopy experiments62 and force curve simulations.51,52,63 The forces acting on the pulling molecules were random due to the thermal noise when the amino acids were in the bulk water region, which implied that there was no obvious trap in bulk water during the movement. These forces were recorded as a function of the distance between amino acids and HAP faces. The departure forces of Glu from (001) and (100) were 2.73 ( 0.27 and 2.12 ( 0.18 nN, respectively. The forces of Gly from (001) and (100) were 1.53 ( 0.08 and 1.81 ( 0.09 nN, respectively. It was noted that the departure forces of Glu were larger than those of Gly because the whole Glu molecule was negatively charged and that of Gly was neutral. These departure forces actually reflect the adsorption strengths of the amino acids at the active binding sites on the HAP surfaces. The larger departure force indicated the higher affinity of the foreign molecules. The weaker affinity of Gly on the HAP crystal was also supported by the crystal growth rate of HAP under different controls of amino acids.15 Here, it was noted that the difference of the departure force for Glu on the HAP (001) and (100) faces (0.61 nN) was larger than that of Gly (0.28 nN). A higher affinity was found for Glu on the HAP (001) face than (62) Fisher, T. E.; Marszalek, P. E.; Fernandez, J. M. Nat. Struct. Biol. 2000, 7, 719. (63) Lim, R.; Li, S. F. Y.; O’Shea, S. J. Langmuir 2002, 18, 6116.

Figure 4. Local density profiles of water (top), sodium ions (solid line, bottom), and Glu (dotted line, bottom) near the HAP (001) interface at different surface coverages of 0/, 0.25/, 0.50/, and 2.25/ nm2, respectively. Peak positions of the water density profile of the control system are marked, and some density profiles are shifted or reverted (for Glu and sodium ions). Left region is bulk water, and right one is the HAP (001) face.

that for the (100) face, while the affinity was higher for Gly on HAP (100) than that for (001). The PMF curves (Figure 11) indicated that the free energy difference of Glu in surface and bulk for HAP (001) and (100) was -406 ( 65 and -326 ( 29 kJ/mol, respectively. The larger free energy difference meant that Glu prefers to adsorb on the HAP (001) face, which was consistent with the higher departure force mentioned previously. The free energy difference of Gly in the surface and bulk for HAP (001) and (100) was -218 (

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Figure 5. Simulation snapshot for adsorbed Glu on the HAP (001) face in water with a surface coverage of 2.25/nm2 and group based local density profile. (A) Final configuration in side view. Amino and carboxylate groups are in blue (nitrogen) and red (oxygen), respectively. Amino groups mainly occupy the vacant calcium sites. (B) Typical adsorption sites of Glu and sodium ions on the interface. For clarity, water molecules are not shown. Sodium ions occupy the vacant calcium sites. (C) Group based density profiles of amino (N), side chain carbonate (CD), and carboxylate (C) groups near the HAP interface. (D) Adsorption sites of Glu on the HAP (001) face (top view). Lineby-line-type pattern, marked by light-green ribbon ([010] direction), is formed by adsorbed Glu on the HAP (001) face.

Figure 6. Local density profiles of water (top) and Gly (bottom) near the HAP (100) interface at different surface coverages of 0/, 0.08/, 0.32/, and 3.20/nm2, respectively. Peak positions of the water density profile of the control system are marked, and some density profiles are shifted or reverted (for Gly). Left region is bulk water, and right one is the HAP (100) face.

29 and -228 ( 33 kJ/mol, respectively, which meant that the affinity of Gly on HAP (001) and (100) was similar. The free energy difference of Gly on HAP (100) was a little bit higher than that of the (001) face, which also was consistent with the conclusion of departure force probing. From the free energy difference point of view, the absorbability of Glu on the HAP faces was also stronger than that of Gly.

4.3. Control of HAP Morphology. When amino acids were strongly adsorbed on specific sites of the HAP surface, they blocked the attachment of the ions of calcium, phosphate, and hydroxyl onto the crystal face. In the solutions, the crystal surfaces were not static; molecules were constantly attaching and detaching. Growth from a supersaturated solution occurred because the flux of molecules attached to the crystal surface exceeded the flux of molecules detached from the surface and therefore resulted in the crystal growth. When the amino acids were introduced into the growth system of HAP, prior to when calcium or phosphate ions could reach their corresponding sites on the growing faces, an additional force was required to remove the adsorbed biomolecules. Since the departure force and free energy difference of Glu from the (001) face was much greater than that from the (100) face, it could be understood that the inhibiting ability for Glu on (001) was more significant. Thus, the crystal growth at the c direction was inhibited, and that at the a and b directions was more preferred, resulting in the formation of plate-like HAP. Accordingly, we could conclude that the inhibiting abilities of Gly were poorer than those of Glu since their departure forces and free energy differences from HAP surfaces were much smaller. As the departure forces and free energy differences of Gly from the (001) and (100) faces were similar, the original features of HAP in the absence of any additive, rod-like morphology and the crystal habits were not altered by Gly. On the other hand, it also indicated that the recognizing skill of Gly on the different HAP faces was poor. These suggestions of the computer simulation were confirmed experimentally. HAP crystallites were synthesized in the presence of 10 mM Gly and Glu, respectively. The synthesized crystals were characterized to be HAP by XRD analysis (Figure 12). The

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Figure 7. Simulation snapshots for adsorbed Gly on the HAP (100) face in water with a surface coverage of 3.20/nm2 and group based local density profile. (A) Final configuration in side view. Amino and carboxylate groups are blue (nitrogen) and red (oxygen), respectively. Amino groups mainly adsorb on the hydroxyl column or occupy the vacant Ca1 sites. (B) Typical adsorption sites of Gly on the interface. For clarity, water molecules are not shown. (C) Group based density profiles of amino (N), R-carbonate (CA), and carboxylate (C) groups near the HAP interface. (D) Adsorption sites of Gly on the HAP (100) face (top view). Two-dimensional pattern, marked by light-green ribbon ([013] direction) and dotted orange line ([0-12] direction), is formed by adsorbed Glu on the HAP (001) face.

Figure 8. Local density profiles of water (top), sodium ions (solid line, bottom), and Glu (dotted line, bottom) near the HAP (100) interface at different surface coverages of 0/, 0.16/, 0.48/, and 2.40/ nm2, respectively. Peak positions of the water density profile of the control system are marked, and some density profiles are shifted or reverted (for Glu and sodium ions). Left region is bulk water, and right one is the HAP (100) face.

morphologies and orientations of these solids were examined by TEM. The images show that rod- and plate-like HAP was formed under the control of Gly and Glu, respectively (Figure 13 B,C). It is well-known that the habits of HAP in the absence of any additive are always rod-shaped (Figure 13A) along the c axis. The typical selected area electron diffraction (SAED) of the formed rod-like HAP under the control of Gly is shown in Figure 13B. The patterns implied that the long axis of the rod-like crystals

was in the HAP c direction, the same as the control ones. In the presence of Glu, the obtained HAPs turned plate-like, and the largest face was (001) (Figure 13C), which was revealed by the 6-fold symmetrical diffraction dots in the SAED pattern and the detailed analysis. Thus, under the control of Gly and Glu, the HAP preferred to grow along the c and a/b axes, respectively, in agreement with the conclusions of computer simulations. However, it was also noted that the change in the crystal morphology of the solids also depended on the concentration of additives. In the case of HAP growth with Glu, it might be caused by the different coverage of Glu on the facets of HAP. We have demonstrated that the adsorption ability of Glu on the HAP (001) faces was significantly stronger than that on (100). Thus, their attachment onto the (001) surface was privileged, resulting in the different coverage densities of Glu on the (001) and (100) faces. The coverage ratio on the two facets would be altered under the different experimental conditions according to the Langmuir adsorption behavior.14-17 It is well-known that another important factor in the inhibition of crystal growth is the surface coverage of the additive. Therefore, the retardant effect of the specific HAP faces might be variable under the different concentrations of Glu, resulting in the concentration-dependent morphology changes of the solids. It has been shown that the maximum densities of Glu on the (100) and (001) HAP faces were 2.25 and 2.40 per nm2, respectively. This implied that, at very high concentrations of Glu, the coverage values of these molecules on these two HAP faces were similar. However, at relatively low concentrations, the coverage ratio of (001) to (100) should be much greater than 1.07 (2.40:2.25) since the selective adsorption of Glu onto the (001) face was preferred. As a result, the inhibition effect on the HAP (001) face was more obvious to form a plate-like morphology. However, at high concentrations of Glu, both the attachments of the additives onto the (001) and

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Figure 9. Simulation snapshots for adsorbed Glu on the HAP (100) face in water with a surface coverage of 2.40/nm2 and group based local density profile. (A) Final configuration in side view. Amino and carboxylate groups are marked in blue (nitrogen) and red (oxygen), respectively. Amino groups mainly occupy the hydroxyl column, and carboxylate groups adsorb around the calcium column. (B) Typical adsorption sites of Glu and sodium ions on the interface. For clarity, water molecules are not shown. Sodium ions occupy the sites of vacant calcium ions. (C) Group based density profiles of amino (N), side chain carbonate (CD), and carboxylate (C) groups near the HAP interfaces. Amino groups stay on both sides of the adsorbed layer. (D) Adsorption sites of Glu on the HAP (100) face (top view). Line-by-line-type pattern, marked by light-green ribbon ([012] direction) and orange dotted line ([0-13] direction), is formed by adsorbed Glu on the HAP (100) face.

Figure 10. Force curves for Gly and Glu departure from the HAP (001) and (100) faces. Departure forces are given for the force curves. Pulling velocity is 0.0005 nm/ps, and the spring force constant is 5000 kJ/mol nm2.

(100) faces approached the maximum values, and their coverage ratio was close to 1. Thus, the plate-like feature of the grown HAP was reduced, which was in agreement with the previous experimental results of the influences of Glu on HAP formation at concentrations of >0.1 M.61 In contrast, the attachment skills of Gly onto the two different faces, (001) and (100) of HAP, were similar, and there was no significant selective adsorption effect on a specific surface. Thus, the coverage ratio of Gly on the two faces could remain almost unchanged at different concentration levels. With similar adsorption abilities and coverage, the concentration-dependent morphology change of HAP in the presence of Gly was not so remarkable. It was also

Figure 11. PMF for Gly and Glu departure from the HAP (001) and (100) faces. Dashed lines show their error ranges.

reported that the profiles of the formed HAP with Gly were almost the same despite the additive concentrations.64 All aspects of a crystal, including phase, habit, and growth rate, can be finally determined by interfacial energetic controls. The depths and shapes of the energy minima control the equilibrium crystal morphology. In the AFM studies,65-67 it was suggested that the additives could form an ordered adsorption layer on the facets, lowering the surface free energy through a (64) Gonzalez-McQuire, R.; Chane-Ching, J.-Y.; Vignaud, E.; Lebugle, A.; Mann, S. J. Mater. Chem. 2004, 14, 2277. (65) Teng, H. H.; Dove, P. M.; Orme, C. A.; De Yoreo, J. J. Science 1998, 282, 724. (66) Orme, C. A.; Noy, A.; Wierzbicki, A.; McBride, M. T.; Grantham, M.; Teng, H. H.; Dove, P. M.; De Yoreo, J. J. Nature 2001, 411, 775. (67) De Yoreo, J. J.; Dove, P. M. Science 2004, 306, 1301.

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Figure 12. XRD spectrum of synthesized HAP crystals without additives and in the presence of 10 mM Gly and Glu.

change in the equilibrium activity of solutions contacting those facets. In the present work, we confirmed that Gly and Glu could form structured adsorption patterns on HAP surfaces. Since the densities of Gly and Glu could be enriched spontaneously on the HAP surfaces, they acted as a surfactant to reduce the surface free energies of the HAP (001) and (100) faces. In the case of Glu, its selective adsorption could alter the equilibrium thermodynamics of the growing surfaces, which stabilized the specific facet of (001). This site-specific binding of amino acid residues to the HAP surface is analogous to that occurring at the dislocation steps of calcite.65-67 It should be mentioned that here we had just modeled the ideally perfect HAP solution of amino acid interfaces. The detailed HAP-interface structure was complicated and still was unknown in experiments. The HAP crystal surface might exist as crystal terraces, steps, dislocation, and kinks, etc., but according to atomic force microscopy experiments,11,18 there were tens to several hundreds of nanometer squared HAP crystal terrace areas for HAP in solution. Physically, our simulation work represented the adsorption behavior of amino acids on HAP crystal terraces in a water solution. Another fact that should be emphasized is that quantum effects were not taken into account in conventional MD simulations. Thus, the processes including chemical reactions (bond formation and breaking) such as proton transfer were not considered in this method. It was expected that the application of ab initio methods22 could improve the understanding of the detailed structures of amino acids on the HAP interface. Here, we show that computer simulations provide another important tool for the understanding of adsorption sites and behavior of various amino acids on HAP crystal interfaces. Our simulation results indicate that an acidic amino acid, Glu, was adsorbed much more strongly than that of neutral one, Gly, which coincided with some experimental findings that the acidic residue of dental-related proteins showed a strong interaction with the HAP faces by solid-state NMR dynamic studies.11 Our previous simulation work68 also indicated that an amelogenin-like LRAP peptide adsorbed strongly on the HAP (001) face with carboxylate group grasped on the calcium ion. Besides, the electronically charged Glu had a better recognizing skill to tell the difference between the (001) and the (100) crystal faces, and it could select the (001) face preferentially. (68) Chen, X.; Wang, Q.; Shen, J.; Pan, H.; Wu, T. J. Phys. Chem. C 2007, 111, 1284.

Figure 13. TEM of the HAP crystals synthesized without additives (A) and in the presence of 10 mM Gly (B) and Glu (C). Insets are their SAED patterns.

5. Conclusion Rod- and plate-like HAP crystallites were formed under the control of Gly and Glu, respectively. The SAED images indicated that HAP crystals grow along the c direction for Gly and in the a/b directions for Glu. The molecular dynamics simulations results indicated that Gly and Glu had specific adsorption sites on the

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HAP (001) and (100) faces and that they had certain structural patterns, thus inhibiting the growth of HAP. The inhibiting ability for Glu on (001) was much stronger than that for (100), which was the molecular reason for the formation of plate-like HAP in our experiment. The different departure forces or the surface excess free energy (the difference of the surface and bulk free energy) of the amino acid from the HAP face determined its regulation ability in the crystallization and resulted in the heterogeneous regulation effects on the HAP facets. It is suggested that the departure force or adsorbability for the amino acid molecules, their detailed adsorption sites, and patterns on the HAP faces were very important in understanding the mechanism of biomineralization. However, these data were difficult to detect experimentally. The advanced MD simulations technique could

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provide another pathway to explore the organic-inorganic interface structure, interactions, and roles in biomineralization at the atomic level. Acknowledgment. This research was supported by the National Natural Science Foundation of China (20571064 and 20601023), the Changjiang Scholar Program (R.T.), and Zhejiang University. Supporting Information Available: Force field parameters and simulation results of HAP crystal parameters. This information is available free of charge via the Internet at http://pubs.acs.org. LA700567R