Molecular Dynamics Simulations of Collagen-like Peptide

6086

J. Phys. Chem. C 2007, 111, 6086-6094

Molecular Dynamics Simulations of Collagen-like Peptide Adsorption on Titanium-Based Material Surfaces Susanna Monti* Istituto per i Processi Chimico-Fisici (IPCF-CNR), Area della Ricerca, Via G. Moruzzi 1, I-56124 Pisa, Italy ReceiVed: January 12, 2007; In Final Form: February 24, 2007

Classical molecular dynamics simulations were performed to investigate the early processes taking place in the interface region between titanium dioxide and a type I collagen triple helical segment, rich in hydroxyproline and proline residues, with special focus on intermolecular interactions and conformational stability. Possible binding modes were identified, and the analysis of the structural macroscopic and microscopic properties succeeded in elucidating the behavior of the helical bundle in contact with the TiO2 layer. The stability of the adsorbed molecules increased with the increasing number of coordinated atoms, and side-chain groups seemed to be primarily responsible of the adsorption process. However, a certain degree of disruption of the bundle was observed and quantified, considering the interchain distances and total solvent-exposed surface area. The results were consistent with previous studies and experimental data, which revealed that major changes in collagen conformation took place in water solution.

1. Introduction Mechanical properties such as tensile strength, stiffness, fracture toughness, and resistance are some of the characteristics required by metals and metal alloys to be used as medical implant materials. Although several substances possess these qualities and are widely employed for treating various diseases and injuries, their clinical application can be restricted by the necessity of two other very important factors, corrosion resistance and biocompatibility.1 Titanium and titanium alloys are well-known to have these requisites, which derive mainly from the presence of a thin and strongly adherent oxide layer, 0.5-10.0 nm thick, which passivates these metals when they are in contact with aqueous solutions or water vapors.2 Due to the favorable bone-titanium interfacial interactions, osteointegration is one of the wellestablished properties of titanium implants surfaces.3 Nevertheless, there is an ongoing interest in modifying implant coverings to improve their cell adhesive properties and tissue integration capacity such as to induce acceleration of normal bone healing phenomena.4 Among the different approaches to the surface modification of bone-contacting titanium devices,5 biochemical methods are very promising. These techniques aim at the creation of artificial biomimetic surfaces through the incorporation of bioadhesive motifs from the extracellular matrix proteins. Beside the binding of peptide sequences,6-11 the research has focused on implant precoating with proteins such as elastin, fibronectin, vitronectin, laminin, or collagen, which include multiple binding patterns supporting attachment of various integrin and nonintegrin receptors, located at the cell membrane. Binding activates signaling pathways able to stimulate cell adhesion, migration, proliferation, differentiation, or matrix mineralization.12-17 Among the main proteins of the extracellular matrix, collagen is of special interest. It has successfully been used to modify biomaterial surfaces, in dental surgery as osteogenic and bone * Phone: +39-050-3152520. [email protected].

Fax:

+39-050-3152442.

E-mail:

filling material,18,19 providing a more rapid regeneration of bone defects, and for other medical applications in several types of morphologies.20 The cellular response induced by collagen is mainly mediated through the amino acid sequence Arg-GlyAsp, which is recognized by integrin receptors located at the cell membrane.21,22 A careful analysis of the collagen primary structure revealed the presence of uninterrupted successions of Gly-X-Y triplets and periodic distributions of clustered polar and hydrophobic residues.23 The high content of proline (Pro), hydroxyproline (Hyp), and glycine (Gly) residues and their location along the sequence are responsible for the high conformational stability of collagen molecules, consisting of three extended, left-handed, polyproline II-like conformations, which are supercoiled in a right-handed manner around a common axis.23-27 Triple helix organization leads to the formation of long, rodlike structures, stiff but flexible, about 1.5 nm wide, and over 300 nm long, with globular domains at both ends. Fibrillar collagens (biochemical types I, II, III, V, and XI) are the most abundant and extensively studied both from the experimental and theoretical point of view.28-36 The present study was designed to investigate and describe, using molecular dynamics simulations, the early processes taking place in the interface region between titanium dioxide and a type I collagen triple helical segment, rich in Hyp and Pro residues and not containing charged amino acids, with special focus on intermolecular interactions and conformational stability. The collagen segment chosen for this research is from a region in the triple helix that is devoid of Arg-Gly-Asp triplets and other charged amino acid side chains to meet the objective of verifying if a stable collagen TiO2 complex is probable in the absence of ionic interactions. Taking into account the fact that, according to experimental observations, amino acid-TiO2 surface preferential interactions are carboxylate group-Ti coordinations2 and, as a consequence, that most of the theoretical investigations dealt with this particular aspect, it was interesting to study and understand what kind of arrangements and

10.1021/jp070266t CCC: $37.00 © 2007 American Chemical Society Published on Web 03/29/2007

Collagen-like Peptide Adsorption on Ti Surfaces

J. Phys. Chem. C, Vol. 111, No. 16, 2007 6087

Figure 2. Two views of the THS (space-filling format) upon the TiO2 surface (lines). The bundle can be considered as a cylinder; h1, h2, and h3 are in yellow, green, and cyan, respectively. Surface Ti and O atoms are in magenta and red, respectively. Figure 1. THS amino acid sequence.

interactions took place when uncharged molecules, without carboxyl groups, were placed near a TiO2 surface. Recent computational investigation of ours, a combination of classical and quantum mechanical methodologies, succeeded in elucidating the adsorption mechanism of differently charged peptide systems, in water solution, on the rutile (110) surface.37-39 The behavior of collagen microfibril segments in pure water or in mixed solutions containing formaldehyde or polyphenols was analyzed and explained in detail in previous studies of ours, demonstrating, through comparison and agreement with experimental results, that the developed force-field parameters and simulation procedures were reliable to describe these systems.40-42 The present work extends and combines the methodologies previously set up to examine possible collagen-TiO2 adsorption mechanisms. 2. Materials and Methods 2.1. Model Building. The initial coordinates of the systems were obtained through model building procedures employing Cerius2,43 AMBER9,44 and Sybyl45 programs. A short triple helical segment (THS) made of 21 amino acid residues, was extracted from a three-dimensional computer model of the bovine type I collagen microfibril based on the Smith description46 made available to us by Professor Eleanor M. Brown.28 The collagen type I model was a heterotrimer formed by two R1 helices (h1, h3) plus an R2 helix (h2). The amino acid content of THS is reported in Figure 1. Side-chain ionization states were adjusted on the basis of the pH of the environment (7.4). Peptide sequences were capped appropriately by adding a NH2 group (Nhe) to the C-terminus and a -COCH3 group (Ace) to the N-terminus to avoid spurious end effects. The net charge of the system was 0. The rutile TiO2 (110) nonhydroxylated surface was created by periodic replication of an elementary cell in the x and y directions.37 The layer in contact with the THS and water molecules contained 64 accessible Ti atoms and 192 O atoms (128 bridging and 64 terminal oxygens) and was about 52 and 27 Å in the x and y dimensions, respectively. THS was

placed close to the TiO2 surface, avoiding bad steric and electrostatic interactions, choosing a reciprocal position that allowed the formation of a hydrogen-bond or Ti-coordinated complex (Figure 2). Six configurations of the THS-surface complex, hereafter named C1-C6, were considered (Figure 3). To maximize the interaction of THS with the layer, the bundle was oriented with its axis parallel to the TiO2 plane (Figure 2), and six different starting arrangements were generated by rotating the molecule in about 60° increments around the THS axis (Figure 3). Even though the built models did not exhaust all possible arrangements, they should give a fair representation of probable THS-TiO2 configurations and allow one to discriminate among different adsorption modes. The systems were inserted in rectangular parallelepiped boxes and were solvated with TIP3P47 water molecules, deleting those waters falling within a 2.0 Å radius from the solute (TiO2 surface + THS). The final molecular assemblies were made of TiO2 surface, THS, and about 7800 water molecules. 2.2. Molecular Dynamics Simulation Protocol. Molecular dynamics AMBER9 package was employed to perform the classical atomistic MD simulations. The AMBER all-atom ff99 force field,48 the TIP3P water model, and developed potential parameters for TiO2 molecules were used to describe the interactions between THS, water, and surface atoms. The surface was frozen during both equilibration and production phases (see ref 37 for details). Each molecular system was energy minimized without constraints for 5000 steps using steepest descent and conjugate gradient integrators; then, in order to generate random water positions, a short MD simulation was performed for 100 ps at T ) 700 K, freezing the solute coordinates. The randomized systems were equilibrated at constant volume and temperature (T ) 310 K) for about 200 ps, followed by a constant pressure equilibration to adjust the system density. Berendsen’s thermostat and barostat were used to control temperature and pressure,49 and the bond length was constrained using the SHAKE algorithm.50 Periodic boundary conditions were applied in the x, y, and z directions. The final box size after equilibration was about 52 Å × 27 Å in the plane of the surface and about 108 Å in z direction (Figure 4). The Ewald summation was used to handle long-range electrostatic interac-

6088 J. Phys. Chem. C, Vol. 111, No. 16, 2007

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Figure 3. The six starting configurations of the THS (space-filling format)-TiO2 (lines) complex; h1, h2, and h3 are in yellow, green, and cyan, respectively. Surface Ti and O atoms are in magenta and red, respectively.

production run trajectory was considered as part of the equilibration phase and thus was excluded from the final analysis. The system configurations were saved every 5 ps, and the resulting trajectories were viewed and analyzed. 2.3. Structural Descriptors. The global structural properties of THS were characterized by size and shape in terms of the radius of gyration Rgyr, the ratio of the largest to the smallest principal moment of inertia (Imax/Imin), and the eccentricity (η).

Figure 4. One of the starting configurations. The THS molecule is shown in the stick format and a solid ribbon (CR). Carbon atoms of h1, h2, and h3 are in yellow, green, and cyan, respectively. The surface Ti and O atoms are represented as magenta and red spheres, respectively. The simulation box size and water molecules are displayed.

tions, and a time step of 2 fs was employed. Production simulations of around 6 ns were performed for each system in the canonical ensemble (NVT). The first nanosecond of each

The variation of these parameters reflected the influence of solvent and surface interactions on the shape of the THS, revealing details of its structural changes, and, together with the distances between pairs of centers of mass of the helices (h1, h2, h3) and between the CR atom of the Gly residue in the middle of each chain and the surface, provided valuable insight into the overall packing stability of the helix bundle and its local dynamics at a microscopic level. Additional parameters that have been analyzed to describe structural evolution of the THS were the accessible surface area calculated through the method of Lee and Richards51 and interchain hydrogen bonds, identified when donor-acceptor distances were smaller than 3.5 Å and when the angle formed by hydrogen-donor-acceptor atoms was smaller than 40°. Hydrogen-bond persistency was expressed via their percentage of occupancy (%occ.), which was defined as the number of structures with the hydrogen bond present (nHBi) divided by the total number of sampled conformations (Ntot), which was equal to 1000 (%occ. ) 100‚nHBi/Ntot). The number of hydrogen bonds with percentages of occupancy greater than 10 and 50% were considered and analyzed. A hydrogen bond with a percentage of occupancy equal to 10% means that only 100 structures show that hydrogen bond, whereas when it is detected in 500 conformations, its percentage of occupancy is 50%. 2.4. Adsorption Free Energy. The adsorption free energy was calculated using the probability ratio method.55,56 The probability distribution of finding the CR atom of the Gly residue located in the middle of each chain at a given distance from


J. Phys. Chem. C, Vol. 111, No. 16, 2007 6089

Figure 5. Distance of the CR atoms of the Gly residue located in the middle of each chain from the TiO2 layer as a function of the simulation time for the C2, C4, and C6 systems (from top to bottom).

the TiO2 interface was converted to a free-energy profile using the equation

Pi ∆Gi ) Gi - G0 ) -RT ln P0

(1)

where R is the ideal gas constant, T is the absolute temperature, and Pi and P0 represent the probability densities of the peptide being in two particular intervals of distances from the surface. ∆Gi represent the relative free-energy difference between two different positions. The normalized probability density distribution for each helix was calculated using the equation

Pi )

Ni

∑iNi

∆di

(2)

where Ni is the number of times that a helix was positioned in a given interval of distances during the duration of the simulation and ∆di is the width of the interval that was chosen equal to 0.2 Å. P0 is the normalized probability density of a reference state, defined considering positions far away from the surface, where nonbonded interactions were negligible. After inspection of the distances of all sampled structures, which are reported in Figure 5 for C2, C4, and C6, the value of 16 Å was chosen as the cutoff distance, and the P0 probability was calculated by averaging the Pi values between the cutoff and the maximum

Figure 6. Imax/Imin (top), eccentricity (middle), and radius of gyration (Å) (bottom) as a function of the simulation time for the C1-C6 systems.

distance found. The overall adsorption free energy ∆Gads was then calculated as the weighted sum of the relative free energies as

∆Gads ≈

∑i Pi∆Gi∆di

(3)

3. Results and Discussion 3.1. THS Structure and Dynamics. 3.1.1. Size and Shape. The radius of gyration of C1, C3, C5, and C6 THSs, shown in Figure 6 as a function of the simulation time, lay within the range of 7.1-8.5 Å and have an average value of 7.8 ( 0.2 Å, which was equal to the value of the starting THS conformation, indicating that the complexes were stable and had nearly the same size. In contrast, the range explored in the C2 and C4 models was larger (7.1-9.8 Å), and the average value was 8.4 ( 0.4 Å, indicating a lower packing density of the three helices. More marked oscillations were visible for Rgyr (C2) and Rgyr (C4), which displayed two different trends. Rgyr (C2) gradually increased from about 8.0 to about 9.5 Å. During the first 700 ps, Rgyr (C4) decreased from about 8.3 to about 7.8 Å; oscillation around this value continued for about 4 ns and then, in a distinct jump, increased over the final 300 ps to about 8.7 Å. The C2


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TABLE 1: Statistics of the Distances (in Å) between Pairs of Centers of Mass of the Helices (h1, h2, h3) for the Six Models C1-C6a system C1 C2 C3 C4 C5 C6

hx-hx

avg

sd

min

max

h1-h2 h1-h3 h2-h3 h1-h2 h1-h3 h2-h3 h1-h2 h1-h3 h2-h3 h1-h2 h1-h3 h2-h3 h1-h2 h1-h3 h2-h3 h1-h2 h1-h3 h2-h3

5.8 5.1 8.8 11.9 10.5 10.3 4.7 6.4 8.4 13.0 8.9 14.3 6.9 6.6 9.2 6.0 5.6 8.4

0.3 0.5 0.3 5.8 3.6 0.7 0.7 0.2 0.8 9.1 1.8 12.0 1.1 0.9 1.4 0.6 0.3 0.4

4.7 3.9 7.2 5.0 5.5 7.2 3.2 5.4 6.6 4.7 5.4 2.5 2.8 4.9 7.2 4.5 4.7 7.2

7.1 6.3 9.7 25.8 20.9 12.0 6.3 7.4 10.0 30.7 22.6 31.5 8.0 7.8 11.2 7.8 6.2 10.4

a Average values (avg), standard deviations (sd), and minimum (min) and maximum (max) values are reported. In the starting THS conformation, the three distances were 5.1, 5.6, and 7.2 Å (for h1-h2, h1-h3, and h2-h3, respectively).

THS appeared to constantly expand as time progressed, while more unstable chain arrangements were explored by C4 THS, as confirmed also by the parallel examination of the distances between pairs of chains, reported in Table 1, and of the time evolution of CR surface distances, shown in Figure 5. In both C2 and C4 simulations, the helix packing was considerably disrupted, relative to the starting model, and helices moved away from one another and from the surface. On the contrary, in simulations C1, C3, C5, and C6, the helices remained packed. The starting Imax/Imin and η values were significantly greater than one (4.67) and zero (0.71), respectively, indicating an initial elongated, almost cylindrical shape. During the simulation time, due to the action of the surrounding solvent, the individual helices within the bundle adopted more curved conformations, with respect to the initial model, and were considerably twisted. Their conformational changes were reflected in the overall shape of the bundles, which turn into a great variety of less elongated ellipsoidal shapes, as evidenced in Figure 6 by the fluctuations of both Imax/Imin and η around lower average values. For C1, C3, C5, and C6, the average Imax/Imin was 3.2 ( 0.6, and the average η was 0.57 ( 0.07, whereas for C2, they were 2.6 ( 0.5 and 0.48 ( 0.09, respectively, and for C4, they were 1.9 ( 0.4 and 0.30 ( 0.09, respectively. The fluctuation observed in the time evolution of Imax/Imin and η were more prominent in models C2 and C4 than it was in the other models, and the values of the two parameters tended to those of a spherical object, suggesting a high degree of disruption of the bundle and drift of the constituting chains into the bulk solvent. Notwithstanding, the reported parameters described the THS conformational landscape satisfactorily well; the most direct approach to investigate morphology and spatial organization of the various parts of the models was to look at snapshots of the systems sampled during the production run. Instantaneous snapshots at the beginning of the simulation for C6 and at the end of the simulations for C6, C2, and C4 are shown in Figures 7 and 8 to highlight the salient features of the evolved complexes. 3.1.2. Accessible Surface Area and Interchain Hydrogen Bonds. It was interesting to note how the value of the accessible surface area of the initial compact structure (Figure 1), which

Figure 7. Snapshots at t ) 0 ns for C6 (a) and at t ) 5 ns for C6 (b) and C2 (c), illustrating THS/surface interactions, bundle stability (C6), and bundle disruption (C2). The THS molecule is shown in the stick format. Carbon atoms of h1, h2, and h3 are in yellow, green, and cyan, respectively. The surface Ti and O atoms are represented as magenta and red spheres, respectively. Water molecules are not shown for clarity. OH and CO functional groups of the peptides interact with the surface through a combination of Ti-O coordination and hydrogen-bond interactions, resulting in strong adsorption of the peptide to the TiO2 surface. Hydrogen bonds and coordinations are displayed as dashed green lines.

was ≈1094 Å2 (with a volume of ≈1650 Å3), changed during the various simulation runs. After relaxation of THS in different positions upon the TiO2 surface, expansion of the bundle (C1-C6) was observed, with an average increase by about 160 Å2 in surface area and by about 109 Å3 in volume, due to both the effect of thermal motion and the migration of solvent molecules. During the production phase, smooth oscillations around an average surface area of 1279 ( 24 Å2 and an average volume of 1778 ( 20 Å3 were observed for C1, C3, C5, and C6, indicating that these dynamic assemblies, undergoing fluctuations in helix conformations and orientations, somewhat unfolded but retained the overall morphology of a bundle. Instead, as already observed in Figures 5 and 6, individual helices underwent major fluctuations during the whole simulation time in model C2 or during the last ≈1500 ps of the trajectory in model C4, increasing the total exposed


J. Phys. Chem. C, Vol. 111, No. 16, 2007 6091 TABLE 3: Statistics of the Interchain Hydrogen-Bond Network for the C1-C6 Systems on the Basis of their Type, Named According to the Atom Involveda Total HBs system bb C1 C2 C3 C4 C5 C6

Figure 8. Snapshot at t ) 5 ns for C4, illustrating a weaker adsorption mode and bundle disruption. The OH functional group of one of the Hyp residues in h1 coordinates its oxygen to a Ti atom and interacts through hydrogen bonds with the terminal oxygens of the surface. The THS molecule is shown in the stick format. Carbon atoms of h1, h2, and h3 are in yellow, green, and cyan, respectively. The surface Ti and O atoms are represented as magenta and red spheres, respectively. Water molecules are not shown for clarity. Hydrogen bonds and coordinations are displayed as dashed green lines.

44 24 44 41 47 50

HBs10

HBs50

bs

btC btN bb

bs

btC btN

bb

13 35 22 15 19 6

37 29 22 33 28 32

10 13 20 18 23 9

35 29 10 9 23 18

83 17 0 0 100 0 60 40 100 0 100 0

6 12 12 11 6 12

45 29 60 73 46 64

10 29 10 0 8 9

bs

btC btN 0 0 0 0 0 0

0 0 0 0 0 0

a Backbone-backbone (bb), backbone-side chain (bs), backboneC-terminus (btC) and backbone-N-terminus (btN). HBs10 and HBs50 represent hydrogen bonds with percentages of occupancy g 10 and 50% (for definition, see Section 2.3), respectively. All of the reported quantities are percentages and are normalized with respect to the belonging category.

TABLE 2: Statistics of the Interchain Hydrogen-Bond Network for the C1-C6 Systemsa system

h1-h2

h1-h3

h2-h3

%occ. g 10%

%occ. g 50%

C1 C2 C3 C4 C5 C6

41 47 38 33 56 35

41 41 44 15 25 26

18 12 18 52 19 39

34 41 31 41 41 35

19 0 13 19 16 13

a

The number of hydrogen bonds with a percentage of occupancy (%occ.) greater than 10 and 50% (for definition, see Section 2.3) are shown. All of the reported quantities are percentages.

surface area by about 19 and 16%, respectively, and decreasing the volume by about 9 and 4%, respectively. This behavior can be interpreted in terms of bundle disruption, which causes formation of gaps between the chains or even migration of the chains into the bulk solvent, as it appears from the examination of Figure 5 where helix 2 (C4 model) diffusion is well evidenced by the gradual increase of its distance from the TiO2 layer. The degree of packing stability of the bundle can be scrutinized through the distances between helix pairs (Table 1). These quantities changed little during the whole simulation for C1, C3, C5, and C6, whereas the largest noticeable changes were those regarding the C4 and C2 configurations. To give a more complete picture of the structural packing and mobility of the helices in the various models, the network of hydrogen bonds formed between them and the evolution of the number of solvent molecules within 3.5 Å of all of the atoms in each bundle (first solvation shell) were analyzed in detail. Hydrogen-bond classification is reported in Tables 2 and 3, and the number of waters as a function of the simulation time is shown in Figure 9 for C2, C4, and C6. C1, C3, C5, and C6 had an equal number of interchain hydrogen bonds, while in C4, the number decreased by about 16%, and in C2, which had the smallest number of interchain hydrogen bonds, the reduction with respect to the number found for C1 was about 47%. As it appears from inspection of Table 2, the h1-h2 and h1-h3 pairs had a higher percentage of intermolecular hydrogen bonds than the h2-h3 pair in C1, C2, C3, and C5, where the h1-h2 association was dominant (56%). On the contrary, in C4, the h2-h3 interactions were favored

Figure 9. Number of waters within 3.5 Å of the THS atoms (first solvation shell) as a function of the simulation time for the C2, C4, and C6 systems.

(52%), as well as in C6, where all of the helices were more tightly packed, as confirmed by the more balanced hydrogenbond percentages and by the trend of the evolution of the helix distances from the surface shown in Figure 5. The number of highly persistent hydrogen bonds, that is, those with occupancies greater than 50%, was always lower than 19%, while less persistent interactions (those with occupancies greater than 10%) were never greater than 41%. As far as hydrogen-bonding type is concerned, the classification was based on the participating atom types, namely backbone-backbone (bb), backbone-side chain (bs), backboneC-terminus (btC), and backbone-N-terminus (btN), and their percentages are reported in Table 3. As expected, of the total number of hydrogen bonds identified during the whole simulation time, the highest percentage was of the bb type for all of the models except C2, where the bs type prevailed and no hydrogen bonds with percentages of occupancy greater than 50% were noticed. The well-conserved interchain hydrogen bonds (%occ. g 50%), representing a small fraction of the total population (see Table 2), were prevalently of the bb type, even though, in C4, a relevant percentage of bs interactions was observed. These data suggest that, in C1, C3, C5, and C6, the THS bundle was quite stable; in C2, it disrupted constantly and gradually from the beginning of the simulation, and in C4, a sudden, more marked disruption, with respect to the C2 one, took place toward the end of the simulation, as confirmed by inspection of the number of waters in the first shell as a function of the simulation time displayed in Figure 9. 3.2. THS-Surface Interactions. As already demonstrated in a previous paper of ours37 and in agreement with data found


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Figure 10. Snapshot at t ) 5 ns for C2, illustrating the strong adsorption mode of h2 and bundle disruption. Some waters of the first shell are displayed. The THS molecule is shown in stick-ribbon format. Carbon atoms of h1, h2, and h3 are in yellow, green, and cyan, respectively. The surface Ti and O atoms are represented as magenta and red spheres, respectively. Hydrogen bonds and coordinations are displayed as dashed gray lines.

in the literature,2,52 water molecules had thermodynamically favorable interactions with the surface, and a stable water layer covered the TiO2 interface in each simulated configuration. Waters were observed to associatively adsorb above each of the terminal Ti sites, remaining there essentially immobile within the time frame of the simulation, and to interact with terminal and bridging oxygens of the surface. A snapshot from the C2 simulation evidencing water behavior is shown in Figure 10. However, the presence of peptide molecules in the vicinity of the surface prevented full water coverage due to more favorable interactions between the surface and peptide functional groups. Even though the THS amino acid sequence does not contain acidic residues such as glutamic or aspartic acids, which showed a much stronger affinity for the TiO2 surface than the other amino acids,53,54 peptide chain adsorption was observed within the simulation time. 3.2.1. Adsorption Mode. Although bundle packing is conserved only in some of the models, the chains located near the surface at the beginning of the simulation maintained the attachment until the end of the production run. THSs showed similar adsorption modes preferentially coordinating the sidechain hydroxyl oxygens of Hyp and Ser residues to titanium atoms and forming hydrogen bonding interactions with the terminal oxygen atoms of the surface. These hydrogen bonds were identified in all of the simulated complexes, and the average donor-acceptor distance was 2.8 ( 0.1 Å. Even though hydrogen bonds are weaker than ionic or covalent bonds, their contribution was significant to improve stability and to determine the orientation of the molecules. The average adsorption distance between Ti atoms and the adsorbate oxygens, according to the present calculations, was about 2.1 ( 0.1 Å, with oscillations in the range of 1.8-2.6 Å. In some of the models, the hydroxyl oxygen of both Hyp residues was coordinated to a Ti atom, and also, coordination of the carbonyl oxygen of the Gly residue located near the N-terminus was observed. In h3 of the C5 and C6 systems, the hydroxyl oxygen of both Hyp residues was coordinated to a Ti atom, and the carbonyl oxygen of the Gly residue located near the N-terminus was also coordinated with a Ti atom. This coordination was accompanied by the formation of hydrogen bonds of both the amidic group of Gly (near the N-terminus) and the N-terminus group with terminal oxygens of the surface, strongly bonding the molecule to the TiO2 layer. In contrast, dicoordination or monocoordination (adopted by h1

in the final configuration of the C4 model) resulted in weaker adsorptions. An even stronger attachment was obtained for h2 in the C2 system due to the formation of a tetracoordinated complex involving a Ser side-chain hydroxyl oxygen, a Ser carbonyl oxygen, a Hyp hydroxyl oxygen, and a Gly carbonyl oxygen (Figure 10). Interestingly, the h2 chain (C2) was the closest to the TiO2 layer, laid essentially flat on the surface, and maintained its alignment until the end of the simulation; its conformational changes were very limited and smaller than those found in the other attached helices. Also in this case, hydrogen bonds involving the amidic group of Gly near the N-terminus, the N-terminus group, and surface terminal oxygens were observed. 3.2.2. Adsorption Free Energy. The adsorption free energies for the two different chains h1 (or h3) and h2 on the TiO2 surface, calculated considering all of the sampled configurations for C1-C6, were about -1.18 and -1.25 kcal/mol, respectively. As shown from the data reported, both chains adsorbed on the metal oxide layer and their adsorption were the result of several contributions from different types of nonbonded interactions involving both side-chain and backbone atoms of the helices and both titanium and oxygen atoms of the surface. Multiple coordinations together with hydrogen bonding determined stronger peptide-surface complexes, as it appeared from the comparison of the adsorption free energy of the helical segments in contact with the surface calculated for each model independently. In C1, C3, C4, C5, and C6, h3 or h1 (which have an equal sequence of amino acids) was in contact with the surface, and among them, the strongest adsorption was observed for C1 and C6 (with an average adsorption free energy of about -2.0 kcal/mol), which had the highest number of bonds with the layer (3 O-Ti coordinations + 4 HBs). The weakest h1-TiO2 complex was obtained for C4 (with an average adsorption free energy of about -0.8 kcal/mol), where only one coordination and a hydrogen bond were observed. The h2 chain was coordinated to the titanium dioxide interface only in the C2 model, and as already discussed, it had the highest number of interactions; as a consequence, its adsorption free energy was the most favorable (about -2.5 kcal/mol). 4. Conclusions Simulation results suggest that peptides rich in Hyp residues, in principle, can adsorb onto TiO2 surfaces, and the stability of

Collagen-like Peptide Adsorption on Ti Surfaces the adsorbed molecules increases with an increasing number of coordinated groups. Hydroxyl coordination alone produces weaker adsorptions than mixed hydroxyl + carbonyl coordination due to stronger competition, in the first case, with water activity. More flexible side chains can reorient their functional groups to form a more favorable interaction with the TiO2 layer and, at the same time, to avoid unfavorable arrangement of the backbone with respect to the surface. Side chains seem to be primarily responsible for THS surface adsorption, and the amount of adsorbed peptides and the adsorption affinity appear to be largely dependent on the number of amino acid residues having active functional groups, that is, those forming persistent contacts with the surface.53,54 The increase of the total solventexposed surface area of the THS models is consistent with previous studies of ours,40-42 which revealed, through the comparison of collagen behavior in pure water and in mixed solutions containing cross-linking agents, that major changes in collagen conformation took place when it was surrounded by water molecules only, in agreement with experimental findings and other theoretical calculations.34 Results clearly indicate that the orientation of the collagen-like peptide in the initial model influences the TiO2 interaction and collapse in the triple helical structure. In fact, the complete hydrogen-bonding network, which stabilizes the triple helical conformation, is highly dynamic and transient. In contrast with the adsorbed chain, which is strongly bonded to the surface and only slightly influenced by water activity, the two flanking chains, far from the TiO2 layer, have weaker interactions with the surface due to screening effects. These chains are more exposed to the action of the solvent, and the only constraints to their conformation and orientation are the interchain hydrogen-bonding patterns. When some of the backbone atoms of the adsorbed chain point to the surface and contribute to peptide anchoring, they are no longer available for hydrogen-bonding interactions with the nearby helices. Thus, the not-adsorbed segments are more easily solvated, and the degree of disruption is higher (as observed in C2). The modeling results support and reinforce experimental suggestions that it is necessary, in order to obtain a reasonable stabilization of collagen molecules, to treat them with chemical cross-linkers and, in order to extend collagen permanence onto titanium-based implant surfaces, to link it covalently to the interface, thus favoring the enhancement in biocompatibility.57-59 Acknowledgment. The author is grateful to Eleanor M. Brown for allowing the use of her collagen structure model and to David A. Case for granting the use of the AMBER 9 package. The author is also thankful to Nanda Lombardi for fruitful discussions. Most of the calculations reported in this paper were carried out on the resources of the CINECA Supercomputer Center (Progetti Supercalcolo 2006 - Fisica della Materia). References and Notes (1) Zitter, H.; Plenk, J. H. J. Biomed. Mater. Res. 1987, 21, 881-896. (2) Diebold, U. Surf. Sci. Rep. 2003, 48, 53-229. (3) Davies, J. E.; Lowenberg, B.; Shiga, A. J. Biomed. Mater. Res. 1990, 24, 1289-1306. (4) Davies, J. E., Ed. Bone Engineering; Em Squared, Inc.: Toronto, 2000. (5) Puleo, D. A.; Nancy, A. Biomaterials 1999, 20, 2311-2321. (6) Rezania, A.; Thomas, C.; Branger, A. B.; Waters, C. M.; Healy, K. E. J. Biomed. Mater. Res. 1997, 37, 9-19. (7) Delforge, D.; Gillon, B.; Art, M.; Dewelle, J.; Raes, M.; Remacle, J. Lett. Pept. Sci. 1998, 5, 87-91. (8) Roberts, C.; Chen, C. S.; Mrksich, M.; Martchonok, V.; Ingber, D. E.; Whitesides, G. M. J. Am. Chem. Soc. 1998, 120, 6548-6555. (9) Kantlehner, M.; Fisinger, D.; Meyer, J.; Schaffner, P.; Jonczyk, A.; Diefenbach, B.; Nies, B.; Kessler, H. Angew. Chem. 1999, 111, 587590.

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Molecular Dynamics Simulations of Collagen-like Peptide

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