Peptide−TiO2 Interaction in Aqueous Solution: Conformational

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Peptide-TiO2 Interaction in Aqueous Solution: Conformational Dynamics of RGD Using Different Water Models Chunya Wu,*,† Mingjun Chen,† Chuangqiang Guo,‡ Xin Zhao,† and Changsong Yuan† Center for Precision Engineering, Harbin Institute of Technology, P.O. Box 413, Harbin 150001, China and State Key Laboratory of Robotics and System, Harbin Institute of Technology, Harbin 150001, China ReceiVed: NoVember 17, 2009; ReVised Manuscript ReceiVed: January 29, 2010

Adsorption behavior and dynamics of Arg-Gly-Asp (RGD) tripeptides with different orientations onto the rutile TiO2 (110) surface in water solution were systematically investigated by molecular dynamics (MD) simulations, using two different water models, TIP3P (transferable intermolecular potential 3P) and SPC/E (extended simple point charge). Possible RGD-TiO2 binding modes in the form of hydrogen-bonding interactions, involving the amide groups (NH3+ or NH2+, NH2) and surface oxygen atoms were identified. The behavior of RGD in contact with the TiO2 layer was elucidated in detail by the analysis of atom-atom distances, backbone dihedral angles and hydration layers distributed over the interface. The simulation results suggest that, the attachment modes of tripeptides with the same starting arrangement are similar when solvated in TIP3P and SPC/E water, but the conformational stability of the amino sequence is somewhat sensitive to the adopted solvent model. Moreover, the intensity of peptide-surface interaction varies widely depending on the initial arrangement of the RGD sequence. 1. Introduction The high corrosion resistance and excellent biocompatibility of titanium and titanium alloys mainly derive from the presence of a thin but strongly adherent oxide layer1 with a thickness of 0.5-10.0 nm. As a result of the insulation effect, deriving from a dielectric constant of the oxide layer approximate to the water, the Ti-implant is no longer regarded as a heterogeneous body by the bone or tissue. To further improve cell adhesive property and tissue integration capacity of Ti-implant, the creation of artificial biomimetic surfaces through the incorporation of bioadhesive motifs from the extracellular matrix (ECM) proteins is a very promising approach. When the Ti-implant is suddenly placed in a biological milieu containing cells, integrin receptors located at the cell membrane will actively search for the specific ligands to bind with the surface. If the ligands exist on the surface with a defined density, keeping certain conformations to expose the ligand-receptor binding contacts, good cellimplant response may be induced successfully.2 Hence, the key to cell acceptance of biomaterial as a homologue is the existence of ligands on the surface, such as fibronectin, vitronectin, laminin, or collagen, but the cellular response induced by these ECM proteins is mainly through the amino sequence Arg-GlyAsp (RGD), which has a high specificity for integrin receptors.3 Although the responsibility of RGD for cell attachment has already been extensively documented since Pierschbacher and Ruoslahti laid the foundation in the mid-1980s,4,5 the true nature of adsorption reaction and adsorption stability between RGD and the outmost oxide layer of Ti-implant, that is, TiO2, should be of special interest in the seeking for surface modification of the bone-contacting titanium devices. * To whom correspondence should be addressed. Tel.: +86(0)45186403252. Fax: +86(0)451-86415244. E-mail: [email protected]. † Center for Precision Engineering. ‡ State Key Laboratory of Robotics and System. § E-mail: (M.C.) [email protected]; (C.G.) [email protected]; (X.Z.) [email protected]; (C.Y.) [email protected].

It is well-known that water is the essential component of blood and tissue fluid.6 Molecular dynamics (MD) simulations of biological systems put special demands on the choice of water model, due to the influence of water on the conformations of solvated biomolecules. Besides adequate reproduction of relevant bulk properties, the requirement of computational feasibility must be absolutely satisfied according to the problem at hand when choosing an appropriate water model. In general biosimulations, a large number of solvating water molecules are often needed to keep the solute (e.g., protein) in its biologically active state-the addition of just one interaction site to an existing water model can lead to a ∼50% increment in the simulation time,7 therefore, the simple three-site rigid, nonpolarizable water models, especially TIP3P8 and SPC/E,9 are still the best candidates in the present-day biosimulations. A type I collagen triple helical segment, rich in hydroxyproline and proline residues, was solvated with 7800 TIP3P water molecules to inspect the early reaction process on the mineral surface by Monti.10 The theoretical analysis for adsorption modes of alanine-glutamine and alanine-lysine short peptides onto TiO2 surface in water solution sponsored by Carravetta11 also adopted the TIP3P water model. And as early as 1994, Smith12 performed a 1.4 ns MD simulation of BPTI in an explicit solvent environment of SPC/E water to calculate the static and frequency-dependent dielectric constant of the protein. Subsequently, Lo¨ffler13 presented a rigorous derivation of linear response theory to calculate the frequency-dependent dielectric properties of the protein/water/ions system and applied it to MD simulations of an HIV1 zinc finger peptide in a box of SPC/E water molecules. Prˇedota14 developed a force field to simulate the interfacial structure of rutile surfaces in contact with SPC/E water-containing dissolved ions. As to the aqueous solution in our biosimulations, both TIP3P and SPC/E models were employed to present the structural and thermodynamical properties of water molecules. The present study was designed to describe the early adsorption process, taking place in the RGD-TiO2 interface

10.1021/jp9109223  2010 American Chemical Society Published on Web 03/17/2010

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Figure 1. RGD tripeptide, color codes: oxygen, red; hydrogen, white; nitrogen, blue; carbon, dark gray.

region, with special focus on the effect of different water models and different starting arrangements of RGDs on the peptide-TiO2 binding modes and conformational stability. Two groups of MD simulations using identical protocols were performed via the Larger-scale Atomic/Molecular Massively Parallel Simulator to simulate the adsorption behaviors of RGDs with various initial orientations onto rutile TiO2 (110) surfaces in TIP3P and SPC/E water, respectively. In addition, the distribution and dynamics of hydration layers in the interface region are also analyzed in detail. 2. Materials and Methods 2.1. Model Building. The ideal (110) surface is the most stable crystal face of rutile TiO2 and its elementary cell (1 × 1) has a dimension of 2a × c, corresponding to 6.497 and 2.958 Å along [1j10] and [001] directions, respectively.15,16 In the present study, the nonhydroxylated rutile surface was created by period replication of the elementary cell in both x- and ydirections, leading to a MD simulation box whose xy-size was 53.24 × 64.97 Å2. Sixteen TiO2 layers, including 5760 Ti and 11520 O atoms, comprised the whole substrate. In the context, surface bridging oxygen atoms are defined as surface oxygen (Os), while surface 5-fold titanium atoms are defined as surface titanium (Tis). RGD tripeptide, terminated with a positively charged residue (Arg) and a negtively charged residue (Asp) (Figure 1), was extracted from a three-dimensional model of the cell adhesion module of fibronectin (FNIII10) made available to us by Dickinson.17 RGD was initially placed close to the surface to avoid bad steric and electrostatic interactions with the lowermost point at a distance of 4 Å from the top layer of TiO2, which was a bit larger than the required distance of direct peptide-surface bonding. Six different starting arrangements were generated by rotating the RGD molecule in about 60° increments around the y-axis, as Figure 2 shown. And hereafter the six configurations of the RGD-surface complex were named S1-S6. Although the models considered did not exhaust all possible arrangements, a fair representation of probable RGD-TiO2 configurations was given to allow one to discriminate among different adsorption modes. The geometries of TIP3P and SPC/E water monomer, the Lennard-Jones parameters and partial charges on atoms are given in Table 1. Models I-VI were constructed in sequence by solvating S1-S6 with 3452 TIP3P water molecules, respectively, while in models VII-IX, S2-S4 were solvated with equal number of SPC/E water molecules. The water molecules falling within a 2.0 Å radius from the RGD were removed (the initial distances of water molecules from the TiO2 surface were far more than 2.0 Å).

Figure 2. The six starting arrangements of RGD-TiO2 complex. TiO2 and RGD are shown by line mode and CPK mode, respectively. Color codes: Arg, green; Gly, blue; Asp, yellow; titanium, light gray; surface oxygen, red.

TABLE 1: Monomer Geometry, Lennard-Jones Parameters, and Partial Charges Describing Water-Water Interactions type\item TIP3P SPC/E

qH (e)

qO (e)

+0.417 -0.834 +0.4238 -0.8476

εO (kcal · mol-1) σO (Å) rOH (Å) 0.1520 0.1554

3.151 3.166

0.9572 1.00

2.2. Molecular Dynamics Simulation Protocol. The simulations of RGD-rutile aqueous solution systems were carried out at T ) 310.15 K in canonical ensemble (NVT) using the Nose-Hoover thermostat18 with a time step of 1 fs. Periodic boundary conditions were applied in the x- and y-directions with a periodic vacuum gap of 3-fold slab model thickness along z-direction. TiO2 was reproduced through Matsui and Akaogi parametrization,19 and the peptide structure was described by AMBER force field.20 Simultaneously, the SHAKE algorithm21 was adopted to constrain all bonds connected to H atoms and the ∠HOH angle with the particle-particle particle-mesh (PPPM) solver22 handling the long-rang electrostatic interactions. The two bottom TiO2 layers were frozen during the whole simulation time. The water solvent of each molecular assemble was initially energy minimized with the RGD and TiO2 surface fixed to remove the abnormal VDW interactions, and then relaxed at constant temperature and volume over 50 ps to randomize the positions of water molecules. Immediately after, the solutes began to relax with position restraints on the carboxyl oxygen atoms (OCOO-). The constraints were removed after 150 ps, and a long period of RGD deposition was presented. Finally, the resulting configuration of each system without any constraint was equilibrated for 2 ns to allow structural and energetic data collection. Over the past years, researchers were inclined to choose several nanoseconds as the period of simulation due to a variety of factors, and had the adsorption mechanism of adsorbates explored successfully. The duration of Monti’s MD simulation mentioned before was only 6 ns but the modeling results supported experimental suggestions very well.10 Shen23 carried out 1 ns MD runs for FNIII10 adsorption on the hydroxyapatite surface after energy minimization and gained adsorption dynamics of protein on the surface of inorganic materials successfully. Hence, 2 ns is suggested to be a reasonable duration to reveal some important aspects of RGD adsorption. 2.3. Structural and Dynamical Characterizations. The global structural and dynamical properties of RGD and hydration layers were characterized by radial distribution function (RDF), coordination number, property of hydrogen bonds, diffusion coefficient, and orientation order parameters of water molecules.

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2.3.1. RDFs and Coordination Number. RDF reflects the average structure of atoms in a specified group around a central atom, and the calculation strategy has already been documented in a previous literature.24 In this paper, the values of RDFs for different atom pairs were all averaged from the last 200 ps results of each simulation. As a characteristic quantity of static property, the coordination number (ncoor) was obtained based on the integration of RDF7

ncoor ) 4πF0

∫0R r2g(r)dr c

(1)

where g(r) is the RDF for central atom (i)-peripheral atoms (group j) pair, that is, g(rij), Rc is the radial cutoff within which the atoms in group j are considered as coordinated ones of the central atom i, and the value selected in this paper corresponds to the first minimum of g(rij); F0 is the atomic density. 2.3.2. Hydrogen Bond. A hydrogen bond is the attractive interaction of a hydrogen atom with an electronegative atom, such as nitrogen, oxygen, or fluorine, and the hydrogen must be covalently bonded to another electronegative atom to create the bond.25 The latter electronegative atom, to which the hydrogen atom is attached, is a hydrogen bond donor, while the former electronegative atom is a hydrogen bond acceptor, regardless of whether it is bonded to another hydrogen atom or not. To analyze the interactions between water molecules or between RGD and water molecules, the pattern of hydrogen bond was identified, considering the donor-acceptor distance lower than 3.4 Å and the donor-H...acceptor angle larger than 135°. Similar definitions have been used by many other authors.26-28 The number, length, and angle of hydrogen bonds per atom, that is, nHB, LHB, and θHB, were obtained by averaging the values from the last 200 ps of each simulation. 2.3.3. Diffusion Coefficient. To quantify the dynamics of water molecules and identify the mobile and immobile layers, the diffusion constant of a homogeneous isotropic fluid14 was calculated

D ) lim

1d

tf∞ 6 dt

〈[ri(t) - ri(0)]2〉

(2)

where ri(0) and ri(t) denote the position vectors for centroid of water molecule i at time 0 and t, respectively. The item 〈[ri(t) - ri(0)]2〉 is the mean squared deviation (MSD). 2.3.4. Orientation Order Parameters. Angle R, β, and Φ were introduced to describe the orientations of water molecules with respect to the TiO2 surface. Angle R, ranging from -90 to +90°, describes the steric angle of water molecule with the surface by measuring the angle between the surface and the rotational axis of water molecule. A value of +90° corresponds to an orthogonally orientated water molecule with Ow down and both Hw atoms up. β is the tilting angle between the rutile surface and the plane defined by the three atoms of water molecule with 0° corresponding to parallel and 90° to orthogonal orientation of the water molecule with respect to the surface plane. Φ is the angle between the surface normal and the bisector of the ∠HOH angle. The orientation order parameters SΦ(z),29 SR(z), Sβ(z) and water density F(z) as a function of distance from the surface were obtained according to

F(z) )

〈N(z)〉 m V(z) w

(3)

Sφ(z) )

〈

1 N(z)

〉

N(z)

∑ 21 (3 cos2 φi - 1) i)1

SR(z) )

Sβ(z) )

〈 〈

1 N(z) 1 N(z)

〉 ∑ 〉

(4)

N(z)

∑ Ri

(5)

i)1

N(z)

βi

(6)

i)1

where N(z) and V(z) are the number of water molecules and the involved volume at a distance of z ( ∆z (∆z ) 0.05 Å) from the surface. Zero will be used as the default value for SΦ(z), SR(z), and Sβ(z) if no water is found at such a distance. SΦ(z) ) -0.5 corresponds to Φ ) 90°, indicating that the rotational axis of water molecule is parallel to the rutile surface, while SΦ(z) ) 1 corresponds to Φ ) 0°, showing a water rotational axis perpendicular to the surface. SR(z) and Sβ(z) are the average values of angle R and β for the water molecules at a distance of z ( ∆z (∆z ) 0.05 Å), respectively. 3. Results and Discussion At implantation in vivo, the soluble proteins in serum will act on the surface of implant simultaneously, thus the precoating must withstand competitive desorption from these body proteins, as initially described by Vroman.30 In this study, some key factors, affecting the RGD-TiO2 binding mode and conformational stability, were distinguished by means of simulation analysis, which can be regarded as a beginning of surface modification for titanium implant with RGD coating. On the basis that RGD is terminated with both carboxyl groups (COO-) and amino groups (NH3+, NH2+, NH2), the distribution of water molecules and various chemical groups, that is, carboxyl and amino groups, over the surface were quantified to determine what kind of interactions were responsible for RGD adsorption onto the TiO2 surface by means of RDFs and coordination number. To be able to better understand the influence of different water models and starting arrangements of RGD on the adsorbates’ adsorption geometries and therefore the hydrophilic interactions in the simulated system, RGD conformation changes, peptide-surface interactions, diffusivity, and orientations of hydration layers during the simulation time were also investigated. Because of the unfavorable interactions, not all of the structures remained near the surface, and indeed, RGD tripeptides in models IV, VI, and IX escaped into the bulk solvent. Hence, models I-III, V, and VII-VIII were classed as the first series in the following sections, whereas models IV, VI, and IX were classed as the second series. 3.1. RDFs and Coordination Number. The RDF results of Tis-water oxygen (Ow), Os-Ow, amide nitrogen (NNH3+, NNH2+, NNH2)-Os and OCOO--Tis for models in the first series, and the RDF results of Tis-Ow, Tis-water hydrogen (Hw), Os-Ow, and Os-Hw for models in the second series were obtained by averaging the instantaneous values of the last 200 ps MD equilibration stage of the corresponding assemblies (Figure 3, Figures S1 and S2 in the Supporting Information). As a consequence of quite similar trends of RDF items for diverse models, model I is selected as the major object for a detailed description of the adsorption phases of RGD and water molecules. The Tis-Ow RDF exhibits a first sharp peak at ∼1.95 Å with a coordination number of 1.00, which indicates that the coverage

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Figure 3. RDFs of TiO2 surface atoms-water, RGD for model I and model V. (a) Model I, (b) model V.

Figure 4. RDF and coordination number of water more than 12 Å from the TiO2 surface. (a) TIP3P water in model III, (b) SPC/E water in model VIII.

of the first molecularly adsorbed hydration layer in the range 1.75-2.45 Å, is exactly one water molecule per 5-fold Ti. The well-separated lower and broader second peak centered at ∼3.55 Å corresponds to the second layer of water molecules interacting with the first hydration layer and the surface bridging oxygen atoms. The data are in excellent agreement with X-ray experimental findings31 (average positions of 2.1 and 3.8 Å for the first and second peak, respectively) and Carravetta’s calculated results11 (average positions of 2.0 and 3.8 Å for the first and second peak, respectively). The presence of a quite low Tis-Ow RDF amplitude (approximate to 0) between the first and second peak indicates that water molecules are truly adsorbed onto the TiO2 surface without any diffusion. The Os-Ow RDF shows a sharp first neighbor peak at 2.85 Å with a coordination number of 4.18, suggesting that on average, four water molecules simultaneously approach one bridging oxygen atom at hydrogenbond (HB) distances. According to the data listed in Table S1 and Table S2 in the Supporting Information, major differences in TiO2-H2O RDFs and coordination numbers between the first and the second series are nonexistent, which indicates that the adsorption of RGD has no obvious effect on the overall distribution of water layers. To test the possible arrangements and interactions when RGD tripeptides deposit from water solution onto the rutile (110) surfaces with different starting orientations, RDFs of OCOO--Tis, NNH3+-Os, NNH2+-Os and NNH2-Os were presented together for a direct comparison. The OCOO--Tis RDF shows that none of the OCOO- atoms is engaged in a direct interaction with the surface, thus OCOO- atoms should be essentially active for hydrogen bonding with water molecules, which can be confirmed by a larger value of OCOO--Ow coordination number (∼3). The distances from NNH3+ (models I-III, VII-VIII) and NNH2+, NNH2 (model V) to Os are far away from the HB distances suggested by X-ray and neutron diffraction experiments.25 The NNH3+-Os RDF of model V with the first peak centered at 2.70

Å indicates that NH3+ group of S5 binds to the TiO2 surface through hydrogen bonds with Os atoms. RDFs of NNH2+-Os and NNH2-Os for models in the first series, except model V, have well-defined sharp peaks centered at ∼2.85 and ∼2.80 Å, minima at ∼3.50 and ∼3.45 Å, and coordination numbers of ∼1.57 and ∼1.52, respectively, which are close to the values obtained by Song28 (rfmax ) 2.85 Å, rfmin ) 3.64 Å, ncoor ) 1.60 for both NNH2+-Os and NNH2-Os). For pure water, the Ow-Ow RDF calculated between the oxygen atoms of water molecules, which are more than 12 Å away from the substrate surface, together with the coordination number of Ow in the same region is presented in Figure 4. The Ow-Ow RDFs of TIP3P water (model III) and SPC/E water (model VIII) exhibit first sharp peaks at ∼2.75 and ∼2.70 Å, respectively, close to Pekka’s calculation32 using the same models (first maximum positions, 2.77 Å for the TIP3P model and 2.75 Å for the SPC/E model). The coordination numbers of Ow, determined by integration of Ow-Ow RDF up to the first minimum, are 4.92 for the TIP3P water and 4.70 for the SPC/E water, respectively, which are slightly higher than the experimental value of ncoor ) 4.5 given by Soper,33 but tally well with other theoretical results (ncoor ) 4.90 for TIP3P water at 300 K,27 ncoor ) 4.86 for SPC/E water at 298 K34). The differences between these results may be explained in part by the difficulty in determining the exact position of the minimum due to the shallowness of the RDF curve. But the agreement of the Ow-Ow RDF curve with real water at 298 K9 is somewhat better for the SPC/E than for the TIP3P model. 3.2. RGD Conformational Changes. As Kasemo documented,6 water molecules are the first ones to arrive at the TiO2 surface during the adsorption process, and the surface water shell will have a profound influence on the adsorption behavior of RGD that arrives a little later. For models in the first series except model V, NNH2+ and NNH2 atoms of Arg side chain bond to the surface bridging oxygen atoms, while for model V, only

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Figure 5. Adsorption conformations of RGD onto TiO2 surface for model I. (a) 180 ps, (b) 950 ps, (c) 1600 ps. TiO2, RGD, and water molecules are shown by CPK mode, ball-and-stick mode and line mode, respectively. Hydrogen bonds are represented by green dashed lines.

Figure 6. Adsorption conformations of RGD onto TiO2 surface for model V. (a) 250 ps, (b) 1000 ps. TiO2, RGD, and water molecules are shown by CPK mode, ball-and-stick mode and line mode, respectively. Hydrogen bonds are represented by green dashed lines.

Figure 7. Evolutions of amino nitrogens-surface oxygens distances during 2 ns molecular dynamics simulation time. NNH2+ and NNH2 refer to amino nitrogens in model I, NNH3+ refers to N-terminal atom in model V.

NNH3+ atom of N-terminal is the adsorption site. Once bonded to the rutile surfaces, the RGD tripeptides have a propensity to remain there and the RGD-TiO2 binding modes are maintained until the end of the simulation. The adsorption conformations of RGDs in model I and V are shown in Figure 5 and Figure 6. Furthermore, the distances between nitrogen atoms and the corresponding Os atoms for these two models during the 2 ns equilibration stage are shown in Figure 7, where the Os atoms refer to the ones locating within the shortest distances from the corresponding nitrogen atoms, because sometimes the nitrogen atoms simultaneously form hydrogen bonds with more than one Os. The adsorption conformations of RGDs for models in the first series except the above two are displayed in Figure S3 and Figure S4 in the Supporting Information.

RGD in model I binds to the TiO2 surface at about 250 ps through interactions of two amide groups, that is, hydrogen bonds of NNH2+ and NNH2 with the nearby bridging oxygen atoms. Instantaneous interruption of one hydrogen bond takes place due to the conformation adjustment of RGD, but the other HB relationship is well maintained at that moment and the broken one will be resumed immediately. Hence, the mean value of HB number (nHB) is 0.95 for NNH2+-Os, while a larger value of nHB ) 1.01 for NNH2-Os derives from two coexisting hydrogen bonds between NNH2 and Os atoms at some transient times. The mean bond lengths and bond angles are LN-Os ) 2.95 Å, ∠NHOs ) 151.34° for NNH2+-Os; LN-Os ) 2.96 Å, ∠NHOs ) 152.45° for NNH2-Os. The NH3+ group, a typical hydrophilic group, does not form any hydrogen bond with the rutile surface in model I, but interacts actively with the surrounding water, which can be confirmed by ncoor and nHB of NNH3+-Ow. The value of ncoor listed in Table S1 in the Supporting Information is obviously larger than the value of nHB in Table S3 in the Supporting Information, because part of ∠NHOw angles do not fulfill the angle requirement of hydrogen bond, however the distances from NNH3+ to Ow are always within the HB range. RGD in model V orients the NH3+ group toward the rutile surface during the first 100 ps of the equilibration process. The NNH3+ atom is then restricted in its movement during the later stage and remains at a distance of 2.72 Å from Os-3, with the standard deviation of 0.12 Å. The nHB of NNH3+-Os pair is a little smaller than 1 due to the failure of meeting the HB angle requirement, while the nHB of NNH3+-Ow pair reaches 1.11, much smaller than the corresponding item for model I as a consequence of the strong NNH3+-Os interaction. Analyzing the distributions of Φ (C-N-CR-C) and Ψ (N-CR-C-N) backbone dihedral angles (indicated by arrows

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Figure 8. Evolutions of backbone dihedral angles of RGD adsorbed onto rutile (110) surface during 2 ns molecular dynamics simulation time. (a) Model I, (b) model V.

TABLE 2: Interaction Energy (kcal · mol-1) and Interaction Force (nN) RGD-Os-1

RGD-Os-2

model\type

energy

force

energy

force

model model model model model model

-77.89 -39.45 -92.51

-3.10 -2.45 -3.35

-70.45 -49.16 -93.75

-2.95 -2.61 -3.30

-40.09 -100.47

-2.66 -3.59

-51.24 -96.09

-2.70 -3.43

I II III V VII VIII

RGD-Os-3 energy

-96.87

in Figure 1), together with their time evolutions during the 2 ns equilibration stage, will help to obtain more detailed information about the conformational dynamics and flexibility of RGD segment after adsorption. In model I, the dihedral angle Ψ of Gly turns abruptly during the first 250 ps (Figure 8a), resulting from the formation of hydrogen bonds between NH2, NH2+, and Os. The backbone dihedrals keep in equilibration after about 600 ps with a short but sudden change of Gly Ψ, which may derive from a twist beginning with the Gly-Asp amido link. As Figure 5c shown, the Asp residue terminated with active hydrophilic groups (i.e., COO- group) moves freely into the solvent and the NH2, NH2+ groups readjust themselves slightly to form hydrogen bonds with Os. In model V, the distributions of dihedral angles flatten out after dramatic fluctuations of Gly Ψ and Gly Φ during the first 500 ps (Figure 8b), which may originate from the readjustment of RGD segment for a more favorable configuration after adsorption. As to models II and VII, where S2 is solvated in TIP3P and SPC/E water, respectively, the dihedral angle Ψ of Gly changes dramatically at 560 and 740 ps, respectively (Figure S5 in the Supporting Information). The distribution of this dihedral angle in model VII gradually shifts to a gentle phase during the later stage, while a sharp rebound of Gly Ψ from 870 to 1100 ps can be observed in model II. In model III and VIII, S3 is chosen as the common solute with TIP3P and SPC/E water being their respective solvents. Gly Ψ of model III declines sharply at about 220 ps and keeps stable for about 800 ps. Shifts of the Gly Ψ curve toward greater values can be detected during the later period, but they return immediately after very short periods. Gly Ψ and Gly Φ of model VIII turn significantly during the first 300 and 400 ps, respectively, then obvious fluctuations of dihedral angels seem to disappear. On the whole, the backbone dihedrals of solutes solvated in the SPC/E water reach equilibrations much earlier than the same ones in the TIP3P water. 3.3. RGD-Surface Interactions. As demonstrated in previous literature,1,14 water molecules have thermodynamically favorable interactions with the surface, and the peptide-TiO2

force

-3.16

H2O-1-Tis

H2O-2-Os

energy

force

energy

force

-63.15 -63.00 -63.00 -63.01 -64.53 -64.52

-4.69 -4.67 -4.67 -4.67 -4.73 -4.73

-14.38 -14.35 -14.34 -14.38 -12.90 -12.93

-1.02 -1.01 -1.01 -1.02 -1.01 -1.02

interface is covered by stable water layers for each simulated configuration. Waters in the first hydration layer are observed to associatively adsorb above the 5-fold Ti sites, remaining there immobile during the later simulation stage, while the molecules of the second water layer interact actively with the surface oxygens and the first hydration layer. However, in models of the first series, the presence of RGD tripeptide in the vicinity of the surface prevents full coverage of the second water layer due to more favorable interactions between the surface and the peptide functional groups. RGD tripeptides in models of the first series maintain their respective equilibration states until the end of the simulation. Similar adsorption modes of RGD (except model V), preferentially coordinating the side chain of Arg residue to the rutile surface are shown and the HB interactions with the Os are established through NH2+ and NH2 groups. While a different attachment is obtained in model V due to the formation of hydrogen bonds involving NNH3+ and Os. Even though hydrogen bonds are weaker than ionic or covalent bonds, they make great contributions to the improvement of molecular stability and the determination of molecular orientation. Interaction energies and interaction forces of the first two water layers and the RGD molecule with the TiO2 surface atoms, obtained by averaging the last 500 ps simulation results, are listed in Table 2, where H2O-1 and H2O-2 refer to water molecules in the first and the second hydration layers, respectively, but the data in Col 8-11 are mean information for the single pair of H2O-1-Tis and H2O-2-Os. The RGD-Os-3 curves displayed in Figures 9 and 10 correspond to the interaction energy and interaction force for model V, while the other data all refer to the information for model I. The imperceptible fluctuations of H2O-1-Tis and H2O-2-Os curves indicate that the first two hydration layers interact strongly with the surface atoms during the whole 2 ns equilibration stage. The interaction energy of H2O-1-Tis pair is about -63.15 kcal · mol-1, much lager than the value of H2O-2-Os pair (-14.38 kcal · mol-1). Therefore, the molecularly adsorbed first-layer water

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F (g · cm-3)

ncoor

TIP3P

0.972 0.986a 0.994e 0.989 0.998i

4.92 4.90b 4.50f 4.70 4.86j

SPC/E

Figure 9. Evolutions of interaction energies of water and RGD with the surface atoms during 2 ns molecular dynamics simulation time. RGD-Os-3 refers to the information of model V; other items all refer to the corresponding details of model I.

Figure 10. Evolutions of interaction forces of water and RGD with the surface atoms during 2 ns molecular dynamics simulation time. RGD-Os-3 refers to the information of model V; other items all refer to the corresponding details of model I.

shell is absolutely immobile, while the second hydration layer shows a reduced liquidity due to a great influence of the surface atoms. During the first 250 ps, a far distance of RGD from the surface prevents strong interactions between them. As the distance decreases, NNH2+ and NNH2 edge out the adsorbed water molecules and orientate themselves toward Os-1 and Os-2, respectively. However, the curves of interaction energy and interaction force for RGD-Os-1 and RGD-Os-2 fluctuate widely from 250 to 600 ps, which is probable that RGD quests eagerly for a favorable adsorption state by means of structural readjustment. Then interaction energies of RGD-Os-1 and RGD-Os-2 converge to -77.89 and -70.45 kcal · mol-1 with interaction forces of -3.10 and -2.95 nN, respectively, during the later simulation stage. RGD in model V approaches the TiO2 surface quickly, and the RGD-Os-3 interaction energy gradually tends to be a stable value, that is, -96.87 kcal · mol-1, which is a bit larger than the interaction energy of RGD-Os in model I, suggesting a more stable adsorption conformation of RGD. According to the data in Table 2, we can find that when solvated in TIP3P water, RGD tripeptides in model II and V, corresponding to initial orientations S2 and S5, present the weakest and strongest interactions with Os atoms, respectively. As to a RGD tripeptide with the same initial orientation but solvated in different water molecules, the interaction energy and interaction force for the SPC/E water model are higher than those for the TIP3P water model.

nHB 3.14 3.5c 0.5-4.6g 3.59 3.34k

diffusivity (10-5 cm2 · s-1) 6.30 5.19d 5.30h 2.92 2.70l,m 2.40n

a Hess,35 TIP3P at 298 K. b Obst,27 TIP3P at 300 K. c Jorgensen,8 TIP3P at 298 K. d Sadeghi,36 TIP3P at 298 K. e Wagner,37 exptl. f Soper, 33 exptl. g Sciortino,38 minimum lifetimes between 3.95 and 0 ps, respectively. h Wu,7 TIP3P at 298.15 K. i Berendsen,9 SPC/E at 300 K. j Svishchev,34 SPC/E at 298 K. k Kowall,39 SPC. l Smith,40 SPC/E at 298 K. m Berendsen,9 exptl at 305 K. n Berendsen,9 exptl at 300 K.

3.4. Hydration Layers Analysis. To further understand the dynamic behaviors of water molecules, we take the simulation results of TIP3P water in model III and SPC/E water in model VIII for a direct comparison. The detailed information is available in Table 3. The average for z-coordinates of 5-fold Ti atoms in the top crystal layer is selected as the starting point (i.e., z ) 0) in the following analysis. 3.4.1. Water DiffusiWity. Considered separately from other layers, the first hydration layer locating within the z-range of 1.75-2.45 Å above the surface was analyzed first. Taking the output conformation of energy minimization stage as the initial state, we obtained the MSD and the corresponding components for each water molecule in the first layer. As a result of high reactivity and great attraction of the surface 5-fold Ti atoms, the TIP3P water molecules reach the surface at ∼10 ps, while the SPC/E water molecules approach the substrate more quickly and reach the surface at just ∼6 ps. Once adsorbed to the TiO2 surface, both waters keep immobile without further diffusion (Figure S6 in the Supporting Information). An undulation of MSD between 50 and 70 ps can be detected in Figure 11, due to the inward relaxation of the outmost surface 5-fold Ti atoms, which were frozen during the first 50 ps but allowed to relax in the later stage. As Song41 documented, the surface 5-fold Ti atoms relaxed inward, thus the molecularly adsorbed first-layer water moved downward along with them without desorption. After a short period of position adjustment, the first water layer tended to be stable again. Moreover, we averaged the diffusivities of water molecules in bins parallel to the TiO2 surface, to see their dependence on the distance from the surface. If a molecule is found at time t in a different bin than it was at time 0, half of the square displacement will be added to the sums in the initial and terminal bins. Prˇedota14 stated that the water molecules diffuse about 3 Å from the origin during 10 and 20 ps. So we analyzed the slope of MSD in segments of 10 ps to improve the statistical accuracy and selected 2.5 Å, which is comparable with the mentioned diffusion length (3 Å), as the height of the bin to measure the diffusion coefficients. This alleviates the issue of molecules crossing bins, effectively including the partial contribution from the nearest neighboring bins only. Distance dependency is observed in both TIP3P and SPC/E water, and the abscissa of data points in Figure 12 is the distance of the starting layer from the TiO2 surface for each bin. We confirm again that the first water layer is indeed immobile after molecular adsorption for both models. The mobility of the second-layer water is about 26% of the bulk value at T ) 310.15 K for the TIP3P model, but 35% for the SPC/E model. The diffusivity

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Figure 11. MSD of each water molecule in the first-layer hydration above the TiO2 surface during the first 100 ps after energy minimization. (a) TIP3P water in model III, (b) SPC/E water in model VIII.

Figure 12. Diffusion coefficient of water layers calculated in 2.5 Å bins as a function of distance from the surface.

of the SPC/E water grows quickly in layers further from the surface, reaching the bulk value of 2.92 × 10-5 cm2 · s-1 when the layer is more than 12.5 Å away from the surface. Smith40 reported the diffusivity of 2.70 × 10-5 cm2 · s-1 for SPC/E water at room temperature. Berendsen9 provided us with the experimental value of real water diffusivity (2.40 × 10-5 cm2 · s-1 at 300 K, 2.70 × 10-5 cm2 · s-1 at 305 K). As is well-known, water diffusivity increases dramatically with the ambient temperature, thus the diffusivity of SPC/E water layer, 12.5 Å away from the surface in our simulation, is in good agreement with these theoretical and experimental results. While the bulk diffusivity of TIP3P water is more steeply approached with the growth of water-layer separation from the surface. The value of bulk diffusivity (6.30 × 10-5 cm2 · s-1 at 310.15 K) tallies closely

with other calculation results (5.19 × 10-5 cm2 · s-1 at 298 K,36 5.30 × 10-5 cm2 · s-1 at 298.15 K7), but it is much larger than the experimental value of diffusivity for real water. 3.4.2. Water Orientation. To discern the bonding mechanism of water molecules with the TiO2 surface, curves of orientation order parameters and water density for TIP3P and SPC/E water molecules locating within the first 14 Å from the surface were presented in Figure 13. For TIP3P water, the peaks of water density and the plus-minus alternation of SR(z) indicate that there are at least four water layers above the surface. However, the small peak of water density locating in the range of 4-5 Å for TIP3P model is not reproduced by the SPC/E water. Moreover, the alteration tendency of SR(z) for SPC/E water abates obviously when water molecules are more than 6 Å from the surface. First-layer water molecules of both models are rotated and tilted with Ow closer to the surface than Hw in a nearly perpendicular orientation with respect to the surface (TIP3P, Rfirst-layer ) 70.62°, βfirst-layer ) 76.85°; SPC/E, Rfirst-layer ) 71.09°, βfirst-layer ) 76.86°), which can be interpreted in terms of Ti-Ow attraction and the expectation of Hw atoms facing the liquid to form hydrogen bonds with the second-layer water molecules. In the second layer, the orientation of water molecules is reversed, exhibiting an Ow-up and Hw-down conformation. For TIP3P water, R decreases sharply to -34.51° and β is around 68.21°, which accord with Kornherr’s results42 (Rsecond-layer ) -33.20°, βsecond-layer ) 70.00°) perfectly. While for SPC/E water, R approaches -25.82° steeply and β is around 66.76°. These available data indicate that the second-layer water molecules have been engaged in a hydrogen-bonding network with the

Figure 13. Water density and orientation order parameters as a function of distance from the surface. (a) TIP3P water in model III, (b) SPC/E water in model VIII.

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first-layer Ow and the surface Os atoms. At the boundary of the first layer, SΦ(z) decreases from 0.80 to nearly 0 for both water models, and maintains this value for a certain distance due to the existence of a thin anhydrous layer between the first two hydration layers. SΦ(z) of the TIP3P water increases to 0.11 at the entrance of the second layer, again it reduces to -0.06 in the center of the second layer. For SPC/E water, SΦ(z) reduces to -0.17 in the center region of the second layer after a small rising. The difference in SΦ(z) variation trends between two kinds of water models vanishes completely when water molecules separate largely from the TiO2 surface as a consequence of decreased influence from the surface atoms. When z > 12 Å, the water molecules are strongly inclined to distribute randomly, just like the bulk water. Water densities of TIP3P and SPC/E models converge to 0.972 and 0.989 g · cm-3, respectively, in good agreement with some theoretical (0.986 g · cm-3 for TIP3P water,35 0.998 g · cm-3 for SPC/E water9) and experimental (0.994 g · cm-3)37 results from other research groups. 4. Conclusions RGD tripepetides with six different starting orientations were put close to the rutile TiO2 (110) surfaces in aqueous solution. The conformational dynamics of adsorbed molecules and the distribution of hydration layers over the peptide-TiO2 interface were investigated by MD simulations. Water molecules confined to the immediate vicinity of the substrate interact with the hydrophilic TiO2 surface actively. The first-layer water molecules associatively adsorb on the surface above each 5-fold Ti site, remaining there immobile during the later simulation stage. While a full coverage of the second water layer, involved in a hydrogen-bonding network with the surface oxygens and the first-layer hydration, is prevented by the presence of RGD tripeptide adjacent to the surface and a reduced liquidity is presented clearly due to the great influence from the surface atoms. But with the growth of separations from the substrate surface, the distribution structure and mobility of hydration layers gradually decay to the liquid properties of bulk water. The simulation results also clearly indicate that the orientation of the RGD tripeptide in the initial model influences the peptide-TiO2 binding mode. RGD in models of the second series escaped into the bulk solvent due to unfavorable interactions, while RGD in model V engaged in hydrogen-bonding interactions with the surface oxygens through NNH3+ atom, however RGD tripeptides in the rest models preferentially coordinated the side chain of Arg residue to the rutile surface, and a different attachment through hydrogen bonds, involving NH2+, NH2 groups and Os atoms, was obtained. Identifying the interaction energy, which may quantitatively evaluate the strength of interaction between a RGD and the rutile surface, we can infer that a RGD tripeptide with the initial orientation S5 presents the strongest interactions with Os atoms, followed by S3 and S1, with S2 coming last. Comparing the behaviors of RGDs in TIP3P and SPC/E water, we find that the adsorption mode of the tripeptide in aqueous solution is similar for these two water models with the main differences in conformational stability of RGD. The trajectories of backbone dihedral angles, as well as the interaction energies and interaction forces between RGD and Os, have revealed that although RGD tripeptides in the first series maintain their respective binding modes until the end of the simulation, the solutes solvated in the SPC/E water reach equilibration much earlier than in the TIP3P water, and the RGD-Os interactions for the former water model are also stronger than those for the latter water model.

Wu et al. The modeling results may still be an approximation to the true properties of RGD tripeptide, but the simulations reported here will help to extend RGD permanence onto titanium-based implant surfaces, thus favoring the enhancement in biocompatibility, which appears attractive to improve cell-implant integration and reduce adverse reactions. Acknowledgment. This work was supported by the New Century Elitist Supporting Program Foundation by the Ministry of Education of China (No. NCET-06-0332) and the 111 project (No. B07018). Supporting Information Available: First peak positions in RDFs and the corresponding coordination numbers, numbers, lengths, and angles of hydrogen bonds for the first series, RDFs of TiO2 surface atoms-water, RGD for model II, III, VII, and VIII, RDFs of TiO2 surface atoms-water for model IV, VI, and IX, adsorption conformations of RGD onto TiO2 surface for model II, III, VII, and VIII at 2 ns, evolutions of backbone dihedral angles of RGD adsorbed onto rutile (110) surface for model II, III, VII, and VIII during 2 ns molecular dynamics simulation time, MSD of each water molecule in the first-layer hydration above the TiO2 surface during the whole molecular dynamics simulation time after energy minimization. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Diebold, U. The Surface Science of Titanium Dioxide. Surf. Sci. Rep. 2003, 48, 53–229. (2) Yao, K. D.; Yin, Y. J. Biomaterials Related to Tissue Engineering; Chemical Industry Press: Beijing, 2003. (3) Liu, X. S.; Luo, H. J.; Yang, H. J. Palladin Regulates Cell and Extracellular Matrix Interaction through Maintaining Normal Actin Cytoskeleton Architecture and Stabilizing β1-integrin. Cell. Biochem. 2007, 100, 1288–1300. (4) Pierschbacher, M. D.; Ruoslahti, E. Cell Attachment Activity of Fibronectin Can Be Duplicated by Small Synthetic Fragments of the Molecule. Nature 1984, 309, 30–33. (5) Ruoslahti, E.; Pierschbacher, M. D. New Perspectives in Cell Adhesion: RGD and Integrins. Science 1987, 238, 491–497. (6) Kasemo, B. Biological Surface Science. Surf. Sci. 2002, 500, 656– 677. (7) Wu, Y. J.; Tepper, H. L.; Voth, G. A. Flexible Simple Point-charge Water Model with Improved Liquid-state Properties. J. Chem. Phys. 2006, 124, 1–12. (8) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D. Comparison of Simple Potential Functions for Simulating Liquid Water. J. Chem. Phys. 1983, 79, 926–960. (9) Berendsen, H. J. C.; Grigera, J. R.; Straatsma, T. P. The Missing Term in Effective Pair Potentials. J. Phys. Chem. 1987, 91, 6269–6271. (10) Monti, S. Molecular Dynamics Simulations of Collagen-like Peptide Adsorption on Titanium-based Material Surfaces. J. Phys. Chem. C 2007, 111, 6086–6094. (11) Carravetta, V.; Monti, S. Peptide-TiO2 Surface Interaction in Solution by Ab Initio and Molecular Dynamics Simulations. J. Phys. Chem. B 2006, 110, 6160–6169. (12) Smith, P. E.; Pettitt, B. M. Modeling Solvent in Biomolecular Systems. J. Phys. Chem. 1994, 98, 9700–9711. (13) Lo¨ffler, G.; Schreiber, H.; Steinhauser, O. Calculation of the Dielectric Properties of a Protein and Its Solvent: Theory and a Case Study. J. Mol. Biol. 1997, 270, 520–534. (14) Prˇedota, M; Bandura, A. V; Cummings, P. T. Electric Double Layer at the Rutile (110) Surface. 1. Structure of Surfaces and Interfacial Water from Molecular Dynamics by Use of Ab Initio Potential. J. Phys. Chem. B 2004, 108, 12049–12060. (15) Abrahams, S. C.; Bernstein, J. L. Gutile: Normal Probability Plot Analysis and Accurate Measurement of Crystal Structure. J. Chem. Phys. 1971, 55, 3206–3211. (16) Andersson, S.; Collen, B.; Kuylenstierna, U.; Magneli, A. Phase Analysis Studies on the Titanium-oxygen System. Acta Chem. Scand. 1957, 11, 1641–1652. (17) Dickinson, C. D.; Veerapandian, B.; Dai, X. P. Crystal Structure of the Tenth Type III Cell Adhesion Module of Human Fibronectin. J. Mol. Biol. 1994, 236, 1079–1092.

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