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Peptide/TiO2 Surface Interaction: A Theoretical and Experimental Study on the Structure of Adsorbed ALA-GLU and ALA-LYS S. Monti,† V. Carravetta,*,† C. Battocchio,‡ G. Iucci,‡ and G. Polzonetti‡ Institute of Chemical Physical Processes - CNR, Via Moruzzi 1, 56124 Pisa, Italy, and Department of Physics and unita` INSTM and CISDiC, UniVersity “Roma Tre”, Via della Vasca NaVale, 84 - 00146 Rome, Italy ReceiVed September 25, 2007. In Final Form: December 10, 2007 The adsorption on the TiO2 surface of two dipeptides AE (L-alanine-L-glutamic acid) and AK (L-alanine-L-lysine), that are “building blocks” of the more complex oligopeptide EAK16, has been investigated both theoretically and experimentally. Classical molecular dynamics simulations have been used to study the adsorption of H-Ala-Glu-NH2 and H-Ala-Lys-NH2 dipeptides onto a rutile TiO2 (110) surface in water solution. Several peptide conformers have been considered simultaneously upon the surface. The most probable contact points between the molecules and the surface have been identified. Carbonyl oxygens as well as nitrogen atoms are possible Ti coordination points. Local effects are responsible for adsorption and desorption events. Self-interaction effects can induce molecular reorientations giving less strongly adsorbed species. The chemical structure and composition of thin films of the two dipeptides AE and AK on TiO2 were investigated by XPS (X-ray photoelectron spectroscopy) measurements at both O and N K-edges. Theoretical ab initio calculations (∆SCF) were also performed to simulate the spectra, allowing for a direct comparison between experiment and theory.
Introduction The study of peptide and protein adsorption onto metal surfaces is extremely important in the field of biomaterials, in biotechnology, and in medical implant sectors. Although a wide variety of metals are nowadays used for fracture fixation devices and stents,1 titanium-based materials are still the preferred choice because of their superior biomechanical properties, reduced corrosion, resistance, and biocompatibility. Nevertheless, to ameliorate these qualities, and above all to improve cell adhesive properties and tissue integration capacity, the research has focused on the creation of biomimetic surfaces through the incorporation of bioadhesive motifs.2 Among the molecules of direct relevance to the surface biochemical modification, extracellular matrix proteins, which include multiple binding patterns, have successfully been used.3-6 However, bioactive compounds of lower molecular weight like ionic self-complementary peptides, able to self-assemble, are definitely of great interest. Since the discovery of EAK16-II, which belongs to this class of molecules, by Zhang and co-workers,7 a number of studies have evidenced their unique characteristics, demonstrating that they support mammalian cell attachment,8 they can be used as scaffolds for neurite outgrowth and synapse formation,9 they form stable † ‡
Institute of Chemical Physical Processes - CNR. University “Roma Tre”.
(1) Disegi, J. A.; Eschbach, L. Injury 2000, 31, D2. (2) Puleo, D. A.; Nancy, A. Biomaterials 1999, 20, 2311. (3) Garcia, A. J.; Reyes, C. D. J. Dent. Res. 2005, 84, 407. (4) Park, J. C.; Kim, H. M.; Ko, J. S. Int. J. Oral Maxillofac. Implants 1998, 13, 826. (5) Tsuchiya, K.; Chen, G.; Ushida, T.; Matsuno, T.; Tateishi, T. Mater. Sci. Eng. 2001, C17, 79 (6) Salasznyk, R. M.; Williams, W. A.; Boskey, A.; Batorsky, A.; Plopper, G. E. J. Biomed. Biotechnol. 2004, 1, 24. (7) Zhang, S.; Holmes, T. C.; Lockshin, C.; Rich, A. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 3334. (8) Zhang, S.; Holmes, T. C.; Dipersio, C. M.; Hynes, R. O.; Su, X.; Rich, A. Biomaterials 1994, 16, 1385. (9) Holmes, T. C.; Delacalle, S.; Su, X.; Rich, A.; Zhang, S. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 6728.
β-sheet microstructures and macroscopic membranes,7,10 and they can form fibrillar assemblies11 similar to those exhibited by β-amyloid. In order to understand the complex interactions that rule the formation of EAK-II peptide TiO2 surface assemblies and to help with the interpretation of experimental data on these complex systems, a preliminary, theoretical, and experimental investigation of the adsorption properties of simpler non-self-assembling H-AlaGlu-NH2 (AE) and H-Ala-Lys-NH2 (AK) dipeptide molecules (Figure 1), which are the repeating subunits of the larger EAK-II peptide, was carried out. Taking into account the fact that, according to experimental observations, amino acids easily bind to TiO2 surface through their carboxylate groups,12-14 and as a consequence most of the theoretical investigations have considered this particular binding mechanism only, we were interested in studying and understanding what kind of alternative arrangements and interactions may take place when several conformations of uncharged AE or AK molecules are deposited from a water solution onto a rutile TiO2 (110) surface in random starting orientations. Our present model systems are not intended to represent a common chemical environment for the two dipeptides, in particular regarding the pH of the solution. In fact, an uncharged AK is present only in basic solutions, while an uncharged AE requires an acidic environment. The present work extends the computational methodology developed in a previous study15 to the simulation of multiple adsorption events of uncharged species and investigates how these different binding modes can be correlated to the resulting experimental X-ray photoemission (XPS) spectra at both O and N K-edges. (10) Zhang, S.; Lockshin, C.; Cook, R.; Rich, A. Biopolymers 1994, 34, 663. (11) Hong, Y.; Legge, R. L.; Zhang, S.; Chen, P. Biomacromolecules 2003, 4, 1434. (12) Diebold, U. Surf. Sci. Rep. 2003, 48, 53. (13) Roddick-Lanzillotta, A. D.; McQuillan, A. J. J. Colloid Interface Sci. 1999, 217, 194. (14) Roddick-Lanzillotta, A. D.; McQuillan, A. J. J. Colloid Interface Sci. 2000, 227, 48. (15) Carravetta, V.; Monti, S. J. Phys. Chem. B 2006, 110, 6160.
10.1021/la702956t CCC: $40.75 © 2008 American Chemical Society Published on Web 02/15/2008
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Figure 1. Stick models of H-Ala-Glu-NH2 (AE) and H-Ala-LysNH2 (AK) dipeptides. Surface atoms are shown in ball mode. Color code: carbon, gray; hydrogen, cyan; nitrogen, blue; oxygen, red; titanium, magenta.
Experimental Section A detailed description of the experimental conditions and methodologies for the synthesis and deposition of AE and AK peptides onto a titanium dioxide layer, including the surface preparation protocol, the experimental equipment used for the XPS measurements, and the data analysis, has already been reported in previous papers of ours;16,17 thus, only those aspects strictly related to the present investigation will be discussed in detail. Briefly, a comparative analysis of C1s, N1s, and O1s signals detected for AE and AK films deposited on Au and TiO2 substrates revealed that adsorption on titanium dioxide occurs for both dipeptides without substantial changes in their chemical structure and the amount of adsorbed peptide is higher in the case of AK molecules. The O1s signals resulting from TiO2 oxygens and chemisorbed and physisorbed waters, identified and discussed in a previous publication,16 were subtracted from the O1s spectra to allow an easier characterization of peptide components.
Molecular Dynamics Simulations The general procedure, including force field parameter determination, surface choice and simulation details have been described in an earlier paper,15 where the model and the computational techniques were also validated. For this reason, only those aspects closely connected with the present work will be discussed. System Setup. The initial configuration of the models for the adsorption simulation were designed using Cerius2,18 AMBER819 and Sybyl20 programs. The rutile TiO2 (110) nonhydroxylated surface, placed in the x-y plane, was created (as already described in ref 15) by periodic replication of an elementary cell in the x and y directions. The layer in contact with peptide and water molecules contained 64 accessible Ti atoms and 192 O atoms (128 bridging and 64 terminal oxygens), and was about 52 Å and 27 Å in the x and y dimensions, respectively. It is well-known12,21 that, different from other TiO2 surfaces,22 the ideal TiO2 (110) surface, the one we consider in the present simulations, represents one of the most controversial cases with regard to molecular versus dissociative adsorption of water molecules. While experiments show a stable adsorbed phase of molecular water at low temperature and indicate that any dissociation at higher temperatures is only mediated by surface defects, some theoretical (16) Polzonetti, G.; Battocchio, C.; Iucci, G.; Dettin, M.; Gambaretto, R.; Di Bello, C.; Carravetta, V. Mater. Sci. Eng. C 2006, 26, 929. (17) Polzonetti, G.; Battocchio, C.; Dettin, M.; Gambaretto, R.; Di Bello, C.; Carravetta, V.; Monti, S.; Iucci, G. Mater. Sci. Eng. C, in press. (18) Inc., A. Cerius2 Modeling EnVironment, release 4.0; Accelrys Inc., San Diego, CA, 1999. (19) Case, D. A. et al. AMBER 8; University of California, San Francisco, 2004. (20) SYBYL Molecular Modelling Software, version 7.2; TRIPOS Associates, St. Louis, MO, 2005. (21) Henderson, M. A. Surf. Sci. Rep. 2002, 46, 1. (22) Ko¨ppen, S.; Langel, W. Surf. Sci. 2006, 600, 2040.
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calculations suggested that dissociation should occur on a defectfree surface too, but a few of them have predicted nondissociative adsorption. We then preferred to consider only nondissociative adsorption in our past and present classical molecular dynamics simulations. Competition of molecular and dissociative water adsorption,23 also in the presence of the dipeptide, will be the subject of the next investigation, adopting an ab initio approach for the simulation. Nine peptide minimum-energy structures, having fully extended conformations, were initially placed close to the surface in different orientations in order to test the stability of diverse adsorption modes in the presence of localized perturbations represented by the neighboring peptide molecules and the water molecules. According to our mentioned interest in investigating alternative arrangements and interactions that may take place when several uncharged AE or AK molecules are present on the rutile TiO2 (110) surface, only one, out of the nine random starting orientations, was adsorbed through its carboxylic group (AE(4)). This particular configuration was largely investigated and the results are reported in a previous paper of ours.15 Although peptides are expected to perturb one another, the main question concerns the range and impact of these perturbations. As already demonstrated in the case of a single peptide,15 both oxygen and nitrogen atoms can favorably interact with Ti sites or be involved in hydrogen bonding interactions with the surface oxygen atoms forming stable complexes. However, in the single peptide simulations the only perturbation was due to the surrounding waters, whose activity prevented a strong peptide surface interaction but did not succeed in causing desorption of the adsorbed molecules. In the present simulations, peptide molecules were initially arranged near the surface choosing reciprocal positions that avoid bad steric and electrostatic interactions and allowed the individuation of plausible assemblages. However, it must be pointed out that the built configuration is just one of the infinite possible arrangements. The two model systems made of the TiO2 surface and the deposited nine AE or AK peptide molecules were solvated with 3000 TIP3P24 waters, energy minimized freezing all surface atoms, and, after a brief molecular dynamics (MD) equilibration stage of 200 ps, subjected to 6 ns NVT-MD production protocol.15
Results and Discussion Peptide Conformational Changes. Peptide-water radial distribution functions (RDFs) indicate25 that all peptide oxygen and nitrogen atoms are involved in hydrogen bonding interactions with water molecules; due to their different distances from the surface, the number of coordinated waters depends on their accessibility. No preferred binding alignment of peptide molecules has been observed in the course of the simulations, and only one desorption event in each system, corresponding to initial orientations (AE(3) and AK(4)), where both carbonyl oxygens were facing the surface at a distance of about 4.7 Å from two titanium atoms and the side chains extended toward the TiO2 layer, took place.25 To characterize the behavior of the adsorbed structures, an extensive analysis of all backbone and side chain arrangements, intramolecular and intermolecular hydrogen bonds, as well as distance of the various groups from the surface has been carried out. Notwithstanding that peptide molecules have considerable flexibility in an aqueous environment, the presence of the TiO2 surface represents a constraint which limits the regions of the conformational space explored by them, especially when (23) Langel, W.; Menken, L. Surf. Sci. 2003, 538, 1. (24) Jorgensen, W. L. J. Am. Chem. Soc. 1981, 103, 335. (25) Monti, S.; Carravetta, V.; Zhang, W.; Yang, J. J. Phys. Chem. C 2007, 111, 7765.
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Figure 2. Final arrangement of AE structures (stick model) bound to the TiO2(110) surface. Surface atoms are represented as gray (titanium) and red (oxygen) balls. Titanium atoms in direct contact with peptide oxygen and nitrogen atoms are green. Peptide carbons, oxygens, nitrogens, and hydrogens are gray, red, blue, and cyan, respectively.
their backbone atoms are in close contact with the surface. The degree of flexibility exhibited by the various structures of both AE and AK sequences along the backbone is strictly connected to the surface coordinated groups. All molecules having more than one backbone contact with the layer, such as structures
AE(8) (Figure 2) and AK(3) (Figure 3), or having just one backbone contact but being involved in intermolecular hydrogen bonds with other peptides strongly bound to the surface, as in the case of structures AE(2) (Figure 2) and AK(7) (Figure 3), remain confined throughout the simulations to single β-sheet or
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Figure 3. Final arrangement of AK structures (stick model) bound to the TiO2(110) surface. Surface atoms are represented as gray (titanium) and red (oxygen) balls. Titanium atoms in direct contact with peptide oxygen and nitrogen atoms are green. Peptide carbons, oxygens, nitrogens, and hydrogens are gray, red, blue, and cyan, respectively.
R-helix regions of the (φ, ψ) (see Figure 1) maps, that correspond to the heavily populated areas of experimental Ramachandran plots.25 On the contrary, molecules more weakly interacting with the surface are less rigid and explore more areas of the map. Structure AK(1) (Figure 3) is a typical example of a flexible adsorbed structure: in its final conformation, the terminus nitrogen
atom of the Ala residue is coordinated to a Ti atom at a distance of 2.1 Å and it is involved in a hydrogen bonding interaction with a nearby surface oxygen. Also, the backbone NH group is hydrogen bonded to an oxygen atom of the surface, but the starting conformation25 was in a quite different three-dimensional arrangement, and major adjustments were necessary to reorient
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Figure 5. Evolution of AE(8) atom-surface distances during molecular dynamics simulation time. Figure 4. Evolution of AK atom-surface distances during molecular dynamics simulation time.
the various groups in order to ameliorate the total interaction of the molecule with the surface and with the adjacent stably adsorbed peptides. Due to unfavorable interactions, not all the structures remained near the surface; indeed, AE(3) and AK(4) molecules25 escaped into the bulk solvent. The examination of atom surface distances during the simulation time was useful to evaluate peptide effective adsorption and mobility of residue side chains under the influence of the different environmental effects (Figure 4). The results showed that atoms in close contact with the surface were restricted in their movements and substantially remained at constant distance (within an oscillation range of about 0.4 Å) from the TiO2 layer, while much greater distance fluctuations were observed for those groups far from the attached portion of the molecule and not associated with other peptide moieties. Remarkable conformational transitions in the side chain structure were caused by local repulsion forces, as observed in the case of AK(7). A striking movement, complete at 3 ns, displaced the NH2 unit in a more favorable position to take advantage of more stabilizing interactions with the surrounding molecules. Even though the interaction with the TiO2 layer suffers from a heavy reduction, the overall balance of the various forces generates a final stable arrangement and sharp jumps are no longer visible in the oscillatory motion of the AK(7) lysine side chain. A noteworthy backbone conformational transition and remarkable molecular reorientation which caused, instead, a lower interaction energy between backbone atoms and the surface, was observed in the case of AE(8) conformation. In the starting spatial arrangement,25 only Ala nitrogen and oxygen atoms were in close proximity to the surface, whereas both the Glu side chain and the NH2 terminus group were far from the layer (more than 6 Å) and could move freely into the solvent. The smooth oscillation of these fragments was maintained for about 5.4 ns, but toward the end of the simulation time, due to adjustments of neighboring peptides and water activity, a sharp reorientation of the backbone led the Glu carbonyl oxygen next to a Ti atom (about 2.1 Å) giving rise to a strong tricoordinated attachment. The evolution of selected AE(8) atom-surface distances is displayed in Figure 5. Surface Binding Mode. Most interestingly, a common pattern emerges for the formation of AE and AK surface bonds in the process of adsorption. This is briefly discussed in the present section; a more detailed report is presented elsewhere.25 60% of adsorbed AE structures are in direct contact with the
surface through their Glu carbonyl oxygen coordinated to a Ti center, while their NH2 groups are involved in hydrogen bonding interactions with the surface oxygen atoms. Only 12% of molecules have more than one coordination point, and a similar percentage (10%) shows the carboxyl moiety in the vicinity of the surface. However, taking into account the fact that the most favorable adsorption geometry for carboxylic acids is a bidentate coordination where the COO- group is directly bonded to two Ti sites, a MD simulation of a “zwitterionic” form of AE, having an unprotonated side chain, was also performed. Two representative structures, used for subsequent XPS spectra simulations, are shown in Figure 6. As far as AK molecules is concerned, of the eight adsorbed conformations with attachment maintained until the end of the simulation, six were bound through coordination of lysine carbonyl oxygen (AK(2), AK(3), AK(5), AK(6), AK(7), AK(9)), while a unidentate coordination was observed for four of these structures (AK(2), AK(6), AK(7), AK(9)). The average adsorption distance between Ti atoms and the adsorbate oxygens of both AE and AK, according to our calculations, was 2.00 Å with a small variation in the range 1.95-2.05, and an average angle between the surface normal and the -CdO vector (γ) of about 35° ( 5° was observed. In some of the AE as well as AK arrangements, the carbonyl oxygens coordination was accompanied by the formation of a hydrogen bond between the terminus -NH2 group and a terminal surface oxygen. Even though hydrogen bonding interactions are weaker than direct coordination to surface atoms, their contribution is significant to improve stability and to determine the orientation of the molecules. In the case of AK peptides, another anchoring group is represented by the amine moiety of the lysine side chain, which could be, in principle, coordinated to a Ti site (not observed in the present simulation) or hydrogen bonded to surface oxygen atoms (AK(6)). The lysine side chain is very long and flexible; it stretches toward the surface at hydrogen bonding distance (from surface oxygen atoms or adsorbed water molecules), interacts with nearby residues, and moves freely into the bulk region of the solvent, as shown by the surface distance plots displayed in Figure 4. Coordination to Ti atoms of both the carbonyl oxygens and of the terminus amide nitrogen, in a tridentate way, has been found to be favorable for three structures, namely, AE(8), AK(3), and AK(5). These molecules were the closest to the surface, and the coordinated O(Ala) atoms were better aligned with the surface normal with respect to the coordinated O(Glu) or O(Lys), having γ ) 23° and an angle fluctuation lower than 3°. Such configurations were very advantageous because they were
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Figure 6. Final arrangement of AEzw(1) and AEzw(2) structures (stick model) bound to the TiO2(110) surface. Surface atoms are represented as gray (titanium) and red (oxygen) balls. Titanium atoms in direct contact with peptide oxygen and nitrogen atoms are green. Peptide carbons, oxygens, nitrogens, and hydrogens are gray, red, blue, and cyan, respectively.
Figure 7. N1s and O1s XPS spectra of AK and AE; curve-fitting components are superimposed to the experimental spectrum.
compatible with the conformational constraints, and the adsorbate internal rearrangement was compensated by an improved linkage to the surface. Interaction with the surface through unidentate nitrogen coordination (AK(1)) or hydrogen bond only (AE(4), AE(5), AE(7), and AK(8)) was responsible for weaker adsorption modes. The results suggest that the balance between optimization
of the local surface-adsorbate interaction and adsorbate structural variations does not play a significant role when deposited molecules are flexible, as in the case of peptides, or have a single contact point to the surface. Simulated and Experimental XPS Spectra. On the basis of their spatial arrangement and more favorable orientation with
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Figure 8. Theoretical simulated XPS spectra of AE in different conformations, from top to bottom: AE(1), AE(2), AE(4), AE(5), AE(6), AE(8), AE(9).
Figure 9. Theoretical simulated XPS spectra of AK in different conformations, from top to bottom: AK(1), AK(2), AK(3), AK(5), AK(6), AK(7), AK(8), AK(9).
respect to the surface, a number of possible conformations of the adsorbed AE and AK species have been selected for the simulation
of the XPS spectra, by ab initio ∆SCF calculations. However, final selection was driven by comparison with experimental
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Table 1. Comparison of Experimental and Shifted Theoretical Ionization Potentials (eV) at the N and O K-edge of AE in Different Adsorption Geometriesa N1s geom.
O1s
exp. (fit)
theo. (shifted)
AE(1)
401.53 401.73 402.58
399.93 401.58 402.03
1.6 0.15 0.55
AE(2)
401.53 401.73 402.58
400.43 401.27 402.05
1.1 0.46 0.53
AE(4)
401.53 401.73 402.58
401.31 401.3 401.84
0.22 0.43 0.74
AE(5)
401.53 401.73 402.58
401.06 402.03 402.38
0.47 -0.3 0.2
AE(6)
401.53 401.73 402.58
401.1 400.95 401.63
0.43 0.78 0.95
AE(8)
401.53 401.73 402.58
399.97 401.06 401.42
1.56 0.67 1.16
AE(9)
401.53 401.73 402.58
399.87 400.71 401.54
1.66 1.02 1.04
AEzw(1)
401.53 401.73 402.58
408.10 402.76 402.26
AEzw(2)
401.53 401.73 402.58
407.93 402.42 402.02
a
exp.-theo.
exp. (fit)
theo. (shifted)
exp.-theo.
531.29 532.18 533.1 536.27 531.29 532.18 533.1 536.27 531.29 532.18 533.1 536.27 531.29 532.18 533.1 536.27 531.29 532.18 533.1 536.27 531.29 532.18 533.1 536.27 531.29 532.18 533.1 536.27 531.29 532.18 533.1 536.27 531.29 532.18 533.1 536.27
531.6 532.01 533.35 535.54 531.43 531.8 533.06 535.66 531.78 532.1 533.34 535.76 532.13 532.49 532.93 535.01 531.5 531.5 532.82 535.19 531.28 531.28 533.98 536.5 531.1 531.1 532.36 534.68 532.09 527.97 527.99 533.23 532.10 528.35 528.24 533.15
-0.31 0.17 -0.25 0.73 -0.14 0.38 0.04 0.61 -0.49 0.08 -0.24 0.51 -0.84 -0.31 0.17 1.26 -0.21 0.68 0.28 1.08 0.01 0.9 -0.88 -0.88 0.19 1.08 0.74 1.59
geom. AE(1)
AE(2)
AE(4)
AE(5)
AE(6)
AE(8)
AE(9)
AEzw(1)
AEzw(2)
The experimental values have been obtained by the fitting procedure described in the text.
findings. The ∆SCF procedure is based on a full separate electronic optimization of the ground state and of the core-hole state. It was chosen because more accurate quantum mechanical methods, such as the Green’s Function (GF) technique, are practically unfeasible for computing core-level ionization potentials of such large molecules as the dipeptides considered here, and because, despite the neglect of correlation effects, this procedure correctly takes into account the relaxation effects that dominate in a core ionization process. Recent investigations have shown that the ∆SCF procedure applied to the interpretation of XPS spectra of amino acids is able to quantitatively reproduce subtle chemical shifts such as those corresponding to different conformers of proline.26,27 Specific effects due to the conformation and orientation of the peptides could not be observed in detail in the experimental XPS spectra; however, by analyzing them through an accurate curve fitting procedure it was possible to discriminate among the sampled conformations those structures contributing significantly to the XPS signals. At the same time, a theoretical analysis was used as a guideline, at a qualitative level, for the interpretation of the N1s and O1s curve-fitted results. All the simulated XPS spectra have been computed for the isolated dipeptide in the adsorbate conformation, i.e., neglecting (26) Plekan, O.; Feyer, V.; Richter, R.; Coreno, M.; de Simone, M.; Prince, K. C.; Carravetta, V. Chem. Phys. Lett. 2007, 442, 429. (27) Plekan, O.; Feyer, V.; Richter, R.; Coreno, M.; de Simone, M.; Prince, K. C.; Carravetta, V. J. Electron Spectrosc. Relat. Phenom. 2007, 156, LXVIIILXVIII.
a direct effect of the substrate and of the intermolecular interactions on the simulated spectra. The quest for ever higher levels of detail and realism in such simulations, as the modeling of “environmental effects” (surface atoms and/or water molecules and/or nearby peptide atoms), requires a huge computational effort. This was considered beyond the purpose of the present first investigation, also in consideration of the limited resolution of the presently available experimental spectra, but we are planning to study the aforementioned effects, hopefully, in the near future. Due to the issue of carbon contamination of the TiO2 surface that contributes strongly to the aliphatic peak (at 285 eV) dominating the experimental spectrum, comparisons between experimental and theoretical data were based only on N1s and O1s orbital energies derived from the different selected conformers. The most significant and representative experimental results are displayed in Figure 7. Theoretical data in Figures 8 and 9 show three distinct groups of ionization potentials (IP) in the ranges 290-296 eV, 404-407 eV, and 535-538 eV associated with the removal of an electron from the C1s, N1s, and O1s orbitals, respectively. It should be noted that the computed ionization energies can be compared to the experimental binding energies after applying a shift of about -4.7 eV, which mostly corresponds to the work function not taken into account by the XPS calculations. According to theoretical results, the N1s ionization energies are grouped into three separate sets which are originated from the different electronic properties of the atoms
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Table 2. Comparison of Experimental and Shifted Theoretical Ionization Potentials (eV) at the N and O K-edge of AK in Different Adsorption Geometriesa N1s
O1s
geom.
exp. (fit)
theo. (shifted)
exp.-theo.
exp. (fit)
theo. (shifted)
AK(1)
399.62 399.83 400.48 400.96 399.62 399.83 400.48 400.96 399.62 399.83 400.48 400.96 399.62 399.83 400.48 400.96 399.62 399.83 400.48 400.96 399.62 399.83 400.48 400.96 399.62 399.83 400.48 400.96 399.62 399.83 400.48 400.96
400.46 400.46 401.22 401.66 400.56 400.56 401.16 401.59 400.32 400.98 400.98 401.46 400.54 400.81 401.5 401.5 399.6 400.95 401.68 401.68 400.45 400.45 401.36 401.7 399.77 400.52 401.43 401.97 400.32 400.32 401.1 402.3
-0.84 -0.63 -0.74 -0.7 -0.94 -0.73 -0.68 -0.63 -0.7 -1.15 -0.5 -0.5 -0.92 -0.98 -1.02 -0.54 0.02 -1.12 -1.2 -0.72 -0.83 -0.62 -0.88 -0.74 -0.15 -0.69 -0.95 -1.01 -0.7 -0.49 -0.62 -1.34
531.78 532.57
531.32 531.44
0.46 1.13
AK(1)
531.78 532.57
531.71 532.11
0.07 0.46
AK(2)
531.78 532.57
531.47 532.11
0.31 0.46
AK(3)
531.78 532.57
531.31 531.4
0.47 1.17
AK(5)
531.78 532.57
531.84 532.28
-0.06 0.29
AK(6)
531.78 532.57
531.26 531.37
0.52 1.2
AK(7)
531.78 532.57
531.77 531.88
0.01 0.69
AK(8)
531.78 532.57
531.29 532.04
0.49 0.53
AK(9)
AK(2)
AK(3)
AK(5)
AK(6)
AK(7)
AK(8)
AK(9)
a
exp.-theo.
geom.
The experimental values have been obtained by the fitting procedure described in the text.
directly bonded to nitrogens. Low ionization energies correspond to the 1s orbitals of the amine nitrogen of the N-terminus (-NH2), whose energy values were spread over 0.5 eV (399.8-400.3 eV) in the case of AE, whereas for AK, the interval was smaller and the IP had roughly a symmetric distribution around the energetic barycentre located at about 400.5 eV. Higher ionization energies, spread in the range 400.7-402.2 eV, corresponded instead to the 1s orbital of the amide nitrogen atoms. This range could be further subdivided into two distinct energy intervals due to the electronic dissimilarity of the two units, bonded to the NH groups. The N1s orbitals with lower ionization energies (in the ranges 401.1-401.5 eV and 400.7401.8 eV for AK and AE, respectively) were those where the NH group was bonded to a carbonyl carbon and to an aliphatic carbon (peptide bond), whereas higher ionization energies, in the range 401.5-402.2 eV, were found for those orbitals associated with the N1s of atoms linked to a carbonyl carbon and to a hydrogen atom. However, the separation between these two regions was not sharp, and a certain degree of mixing was observed in the two systems, which was greater in the case of AE conformers. As far as the amine group of the lysine side chains is concerned, the ionization energies of the N1s orbitals were spread in a narrow energy range of about 0.5 eV centered at about 400.5 eV, which exactly corresponds to the amine nitrogen region. The curve-fitted N1s region for a limited number of selected structures of AE and AK systems are shown in Figure 7, as an example, while the fitting parameters for all the considered structures are reported in Tables 1 and 2. The nitrogen signal was fitted in both cases with three peaks corresponding to the three distinct domains identified. As it appears from the fitting results, the plots were qualitatively comparable with each other in each
system; however, better agreement with the experimental data was observed in the case of AK molecules. Even though the accord was satisfactory, to account for the marked asymmetry of the peak at the higher binding energies an extra contribution coming from peptide molecules with charged side chains is necessary. For this purpose, we computed the O1s and N1s XPS spectra of the two “zwitterionic” forms AEzw(1) and AEzw(2) shown in Figure 6; the corresponding IP values are reported in Table 1. As expected, the chemical shift of the N1s IP of the protonated N terminus moves to the positive side, in agreement with the presence of a weak band at high energy in the experimental spectrum in Figure 7, but its value turns out to be extremely large. This is due to the presence in the model system of a full positive charge (H+) close to the N1s orbital. In order to have a more reasonable picture of the protonated system, we performed the N1s IP calculation for an AEzw molecule surrounded by a small number of water molecules. The presence of the water molecules leads to a more reasonable model where the extra positive charge is now not completely localized around the N nucleus. As a consequence, the computed chemical shift is reduced by about 4 eV, still giving a contribution on the highenergy side of the spectrum, but now in much better agreement with the experimental findings. The origin of the observed highenergy contribution in the N1s XPS spectrum can then be related to the presence in the sample of dipeptide molecules in their zwitterionic form; however, the low intensity of the side band indicates that the fraction of such conformations is quite limited. As far as the O1s region is concerned, theoretical data showed contributions at low ionization energies distributed over a range of about 1.3 eV (531.0-532.3 eV) for both AE and AK systems, that were associated with the two carbonyl groups of the peptide
3214 Langmuir, Vol. 24, No. 7, 2008
backbone. The O1s orbitals corresponding to higher ionization energies were, instead, those of the GLU side chain carbonyl and hydroxyl groups, which were in the ranges 532.4-534.0 eV and 534.5-536.5 eV, respectively, see Figures 8 and 9 and Tables 1 and 2. As it appears in Figure 7 and Tables 1 and 2, the XPS O1s signal was well-fitted with two components in the AK system, while the broader peak displayed by AE molecules was fitted with four peaks corresponding to the identified types. A slightly asymmetric shape of the peak was evident, but the main contributions derived from carbonyl oxygen components. The quality of the AE fits was, however, less satisfactory than the one found for the other investigated system. On the basis of the best fitting results of both N1s and O1s XPS spectra, a number of conformations, namely, AE(2), AK(3), and AK(7), were identified as partially responsible for the detected signals; in fact, also structures AE(4), AE(5), AE(6), AK(1), AK(2), and AK(5), as evidenced by the fitting results, could contribute, perhaps, to a minor degree to the experimental spectra
Conclusions The MD simulations reported in this paper represent, to the best of our knowledge, one of the first investigations of adsorption of multiple peptide conformers on a TiO2 surface, using a classical all-atom methodology. The present work complements a previous study of ours where the model and the computational techniques have been developed and tested. The results suggest that 6 ns is a reasonable duration to explore some important aspects of adsorption and desorption, namely, binding mode, perturbation effects due to neighboring peptides, and solvent activity, and give a detailed description of plausible surface adsorption mechanisms, which happen on a shorter time scale. However,
Monti et al.
we must consider the possibility that significant changes in the whole picture may occur during longer simulation times. The inherent complexity of solvated biomolecules adsorbed on a surface has been recognized in terms of multiple coordination. The confirmation of the most probable contact points between molecule and surface (see ref 25 and references therein) has allowed us to build a coherent structural model of the adsorbed species. Indeed, apart from the well-investigated adsorption by the deprotonated carboxylic group (see ref 15 and references therein), carbonyl oxygens as well as nitrogen atoms are all possible coordination points, and a preferred surface binding mode involving the -CONH2 terminal group has been observed. Depending on local effects, interpeptide interactions can promote or hinder peptide-TiO2 surface attachment, and self-interaction effects can induce molecular reorientations giving rise to less strongly adsorbed species. A comparison of experimental and ab initio simulated XPS spectra provides, despite the limited energy resolution of such spectra, some useful information for selecting the most probable conformational structure of the adsorbates. In particular, for AE a conformation with one backbone (carbonyl group) contact and two hydrogen bonds with the surface, while for AK a conformation with three backbone (two carbonyl and the NH2 groups) contacts with the surface, have been pointed out. Acknowledgment. Most of the calculations reported in this paper were done on the resources of the CINECA Supercomputer Center made available to us through the Progetti Supercalcolo 2006 - Fisica della Materia. This research is supported by a MIUR PRIN-COFIN 2005 research program. LA702956T
