J. Phys. Chem. C 2009, 113, 2433–2442
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Adsorption of Ionic Peptides on Inorganic Supports Susanna Monti,*,† Michele Alderighi,‡ Celia Duce,‡ Roberto Solaro,‡ and Maria Rosaria Tine´‡ Istituto per i Processi Chimico-Fisici (IPCF-CNR), Area della Ricerca, Via G. Moruzzi 1, I-56124 Pisa, Italy, and Dipartimento di Chimica e Chimica Industriale, UniVersita` degli Studi di Pisa, Via Risorgimento 35, I-56126 Pisa, Italy ReceiVed: October 21, 2008; ReVised Manuscript ReceiVed: December 12, 2008
Molecular dynamics (MD) simulations were performed to investigate the adsorption mode of a short threeresidue peptide, namely H-Lys-Glu-Lys-NH2, having only hydrophilic amino acids with alternating negative and positive charges, on titanium dioxide, muscovite mica, and graphite surfaces and to characterize its conformational behavior upon adsorption. In agreement with experimental data, the peptide and its aggregates can weakly adsorb on graphite, and strongly adsorb on both titanium dioxide and muscovite, engaging direct and indirect interactions (mediated by calcium and potassium ions) with the surface atoms through the amino acid side chains. 1. Introduction In recent years research in the field of supramolecular chemistry and molecular recognition has focused its attention on biomolecular systems with the ability to self-assemble and form supramolecular structures of various shapes and definite properties which make them a very viable means in optical, mechanical, electronic, and biomedical applications.1-5 In particular, increasing effort is currently being devoted to creating new hybrid synthetic systems with multifunctional characteristics combining self-assembling peptides and metal, metal alloy, and/or semiconductor solid supports.6-8 However, in order to tailor the structural and physicochemical properties of these inorganic surfaces and impart additional functionalities to the new materials, comprising high biocompatibility, it is essential to gain a detailed knowledge of the adsorption scenario and a clear understanding of how the peptide molecules combine and adsorb onto the solid supports. Extensive experimental studies have shed some light on the behavior of various amino acids, peptides and their aggregates, proteins, and other interesting biomolecular systems deposited on clean surfaces under controlled conditions, whereas only a few theoretical investigations have focused on these classes of compounds.9-17 However, many aspects of the adsorption mechanism and the influence of the interface on the aggregation process are not yet clarified and remain the subject of some ambiguity. The aim of the present work is to provide a detailed description, at the atomic level, of the adsorption behavior of the H-Lys1-Glu2-Lys3-NH2 peptide (from now on one letter code will be used for the amino acids as follows Lys ) K and Glu ) E), recently investigated in our group18 on three different types of layers, namely rutile (1 1 0), muscovite (0 0 1), and highly oriented pyrolytic graphite (HOPG), and give, possibly, new insights in the role played by the solid supports estimating the peptide affinity for the examined materials. The choice of this kind of inorganic substrates was dictated by the fact that titanium-based materials have been extensively employed, for their advantageous mechanical and chemical properties such * Author to whom correspondence should be addressed. Phone: +39050-3152520. FAX +39-050-3152442. E-mail:
[email protected]. † Istituto per i Processi Chimico-Fisici. ‡ Universita` degli Studi di Pisa.
as tensile strength, fracture toughness, corrosion resistance, and biocompatibility,19 in the creation of artificial biomimetic surfaces (obtained through the incorporation of bioadhesive motifs such as oligopeptides and proteins) able to adsorb on medical implants and regulate the biological response to the new material directly influencing implant biocompatibility. On the other hand, muscovite mica and HOPG are a popular choice for the study of interfacial phenomena, the former because its almost perfect cleavage along the (0 0 1) planes removes the complexities introduced by the surface roughness and the latter because its surface is highly ordered and chemically inert. Moreover, all of these supports are used in atomic force microscopy (AFM) measurements to characterize the aggregates, to image the structure of the systems with subnanometer resolution under physiological conditions, and to measure their interaction forces qualitatively. Indeed, AFM has proven to be an appropriate technique to depict different stages of the formation of self-complementary peptide aggregates on various supports20 and to demonstrate their supramolecular organization. The paper is organized as follows. In section 2 the main characteristics of the three studied surfaces are briefly described. The computational procedure is accounted for in section 3, and the results of the various simulations for the adsorption of a small aggregate of the H-KEK-NH2 peptide (Figure 1), made of two hydrogen bonded conformations, extracted from a previous MD study,18 on the three interfaces are reported in section 4. 2. Molecular Models 2.1. Structure of the Supports. MuscoWite Mica. The starting structure used in our calculations was a model of the muscovite (0 0 1) surface, which was kindly provided to us by A. G. Kalinichev21 together with the force field parameters to describe the interatomic interactions (CLAYFF force field22,23). Briefly, the model was built by cleaving the monoclinic C2/c 2M1 muscovite crystal structure, a 2:1 layered dioctahedral aluminosilicate (KAl2 (Si3Al)O10(OH2)), along the (0 0 1) plane at the middle of the interlayer space. One mica (0 0 1) layer consists of one octahedral aluminum sheet sandwiched between two tetrahedral silicon/aluminum sheets, which expose the
10.1021/jp809297c CCC: $40.75 2009 American Chemical Society Published on Web 01/21/2009
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Figure 1. Stick model of the H-Lys1-Glu2-Lys3-NH2 peptide. On the left-hand side the small aggregate of the H-KEK-NH2 peptide used in our MD simulations (made of two hydrogen bonded conformations), is displayed. Inter- and intramolecular hydrogen bonds are shown as green lines.
basal oxygens of the six-member rings. Because of the substitution of Si by Al in the tetrahedral layers (with a ratio Al/Si ) 1/3) the mica (0 0 1) surface has a net negative structural charge, which is balanced by positively charged potassium counterions (K+). K+ ions were located above the Si4Al2 ditrigonal rings.24 The model used in this study is a low energy configuration obtained by MD simulations21 where the surface contains 8 Al, 24 Si, 48 bridging oxygens, and 8 K+ ions. The simulation cell has x and y dimensions of 17.79 Å and 20.73 Å, respectively. Titanium Dioxide. The TiO2 rutile (110) surface is chosen for these investigations because it is one of the most important, extensively studied, and highly stable interfaces. It is composed of a complete Ti-O layer including rows of exposed, 5-fold titanium sites and rows of doubly coordinated bridging oxygen atoms that protrude above the surface and are bound to two 6-fold titanium atoms located in the surface plane. Rows of exposed 3-fold oxygens are also included in the surface plane. The rutile (110) interface derives from the relaxation of the rutile bulk structure cleaved by the (110) plane, and when in contact with water its relaxation at room temperature is minimal and perpendicular to the surface itself.25 The surface unit mesh had dimensions a ) 6.497 Å and b ) 2.959 Å along the rutile [1j10] and [001] crystallographic directions. The adsorption of water on the rutile (110) surface has been investigated extensively both from an experimental and theoretical point of view and even though the extent of water dissociation aroused a lot of controversy between theory and experiment, they agreed in identifying both molecular and dissociative adsorption at room temperature.26-29 Taking into account the fact that both types of surface water attachments can coexist on the perfect TiO2 layer, and that the point of zero charge (pHzpc) of rutile is about 5.4 at 25 °C, a negatively charged partially hydroxylated surface is considered in our simulations.30-32 The negatively charged partially hydroxylated surface was built adding a selected number of terminal hydroxyls as if originating from the neutral nonhydroxylated surface by conversion of some of the water molecules that naturally adsorb above the 5-fold surface Ti atoms to bonded hydroxyls. The number of terminal hydroxyls, which determined the surface charge density, corresponds to a pH value of about 7 and their location corresponds to minimal Coulombic repulsion. The net negative structural charge was balanced with positively Ca2+ counterions. The surface parameters, charges,
and location of the hydroxyl groups have been exhaustively described.30,31 Considering that terminal hydroxyls can be engaged in hydrogen bonding interactions with water and peptide molecules, they were kept flexible during the whole simulation runs, restraining, however, the Ti-O bonds lengths to average values (determined by ab initio calculations30) using a harmonic potential with a force constant equal to 25 kcal mol-1Å-2. The surface structure was built using Cerius2 software33 by periodic replication, in both x and y directions of an elementary cell containing four surface Ti atoms, four surface oxygen atoms, and two bridging oxygens leading to a MD simulation cell about 21 × 21 Å2 dimensions. Highly Oriented Pyrolitic Graphite (HOPG). A bidimensional slab model of R-graphite was built transforming the hexagonal unit cell with lattice parameters a ) b ) 2.46 Å, c ) 6.80 Å, R ) β ) 90°, γ ) 120° into a more convenient monoclinic lattice with a′ ) 2.46 Å, b′ ) 4.26 Å, c′ ) 6.80 Å and R ) β ) γ ) 90°. The unit cell was then replicated to obtain a single layer slab model of 840 carbon atoms with surface area of 37.05 × 59.68 Å2. The ideal C-C bond length was set at the mean value of 1.42 Å. The interaction between the adsorbed molecule and the surface was described, following the method proposed by Drew et al.,34 using a Lennard-Jones potential and point charges located on the carbon atoms and on dummy atoms placed at 0.47 Å above and below the carbon atoms to represent π electrons. The charge assigned to these points was equal to -0.5e and was balanced by a +1e charge of the carbons.34 Considering that the π cloud can be engaged in direct interactions with water and peptide groups, the dummy atoms on the face in contact with the peptide molecules were kept flexible during the whole simulation runs, freezing their distance from the carbon center. 2.2. Adsorbed Peptides. A small H-KEK-NH2 peptide cluster made of two tripeptide molecules was extracted from a previous molecular dynamics simulation run in water solution, which was performed in order to characterize the conformational behavior and self-assembling properties of this sequence. The system consisted of 10 H-KEK-NH2 peptide molecules, 20 charge balancing Cl- counterions and 5512 water molecules. The concentration was about 0.1 M and the box dimensions were 55 Å × 62 Å × 65 Å. The total simulation time was about 11 ns with the first nanosecond counted only as equilibration. The molecules which were randomly dispersed within the simulation box during the whole production run showed the
Adsorption of Ionic Peptides on Inorganic Supports
Figure 2. Atomic density profiles for Ca2+ and K+ ions at both totally and partially hydrated muscovite and TiO2 surfaces.
tendency to evolve into aggregate configurations, that is small clusters mainly made of two tripeptides interacting with each other through their oppositely charged K and E side chains. The sampled dimers had a marked ellipsoidal shape and no preferred spatial arrangement of the constituting monomers, which were organized in a variety of orientations due to their high flexibility in water solution, was observed. The selected cluster has a large amount of surface roughness able to host water molecules and appears to be well solvated. The assemblage was highly dynamic; in fact it constantly formed and dissolved, enabling the monomers to repack and allowing the individual peptides to undergo conformational changes. The cluster was placed on the three surface models. The peptides were quite close to each other, in different orientations, and the location with respect to the interface was chosen on the basis of the nature of the surface and of earlier findings.14,15 According to our previous investigations on amino acids adsorption on TiO214 and to experimental studies,35 amino, carbonyl, and carboxyl groups interact favorably with the surface-forming hydrogen bonds or coordinated complexes. Because of the conformational flexibility of the peptides it was possible to place these binding moieties near the surface atoms (at an average distance of about 2 Å). As demonstrated in earlier simulations,14,15,36 molecular arrangements where these groups have close contacts with the interface are reasonable starting conditions to simulate a real system. 3. Molecular Dynamics Simulations 3.1. Simulation Protocol. The systems, made of the surface and the peptide molecules, already described in sections 2.1-2.2, were inserted in rectangular parallelepiped boxes and solvated with TIP3P37 water molecules removing those waters falling within 2 Å from the surface/peptide complexes. The protonation states of ionizable residues were chosen according to the pKa of the isolated amino acids and a pH of 7.4. The N terminus was considered ionized. As a consequence the H-KEKNH2 peptides had a net positive charge of +2e that had to be neutralized by adding negatively charged counterions (Cl-). All simulations were performed using the AMBER938 package with the ff0339,40 force field to describe the peptides, the CLAYFF parameters for the muscovite surface, and developed parameters for the TiO2 atoms.14,30 All simulations were made with the two peptides on one side of the layer whereas on the other side only water molecules and ions were present. This configuration provided two effectively independent interfaces, which allowed for characterizing the structure of the interfacial solvent and for understanding how it is affected by the presence of the peptide molecules.
J. Phys. Chem. C, Vol. 113, No. 6, 2009 2435 The counterions were added using the standard procedure described in the AMBER9 manual. The command addions in xleap was used. It neutralizes the system which is made of the surface, the peptides, and water molecules by adding selected counterions (Ca2+ or Cl-) in the space around the system using a Coulombic potential on a grid. Before the MD simulations were started, the cell height was adjusted in a series of minimization runs in order to achieve the correct density of the water molecules filling the simulation box and to define an effective three-dimensional periodic system where the interaction between the surface and its z image (slab-slab interactions) was negligible. The final box heights and the corresponding water molecules were 88 Å (1417 waters), 75 Å (674 waters), and 37 Å (2496 waters) for TiO2, muscovite, and graphite, respectively. Then the systems, consisting of the surface, two peptides, counterions, and water molecules, were subjected to constant volume MD at high temperature (T ) 600 K) with solute and surface frozen, in order to randomize water positions. Then preequilibration in the NVT ensemble (T ) 300 K) was performed. Bond lengths were constrained using the SHAKE algorithm,41 and the time step was set to 1 fs. Periodic boundary conditions were applied in x, y, and z directions, and the particle mesh Ewald method was used to deal with electrostatic forces. The surface was kept fixed at the initial geometry (crystal geometry in the case of TiO2 and HOPG) during all the simulations. All details about the employed methodology are described in previous papers.14,15,36 Starting from the last obtained equilibrium configuration, production runs were performed in the NVT ensemble with a total simulation time longer than 20 ns for TiO2 and mica, whereas in the case of HOPG the time span of the simulation was relatively short (8 ns) but long enough to characterize peptide general behavior. 3.2. Simulation Analysis. The location of the peptides with respect to the surface was mapped by calculating the distances of selected peptide atoms from the various layers, and a full characterization of their motion was obtained by means of spatial distribution functions (SDFs), which, in contrast to RDFs, accounted for both radial and angular atom-atom correlations through the visualization of three-dimensional (3D) local atomic densities. The 3D local density profiles were obtained by binning atom positions from rms coordinate fit frames over all the surface atoms at 1 ps intervals into 0.2 Å grids over the whole trajectory. The value of each grid element is the number of times the coordinates of selected atoms are within a specific cubic grid element. In the case of TiO2 the surface plane was defined as the plane containing the exposed 5-fold titanium sites, whereas in the case of muscovite it was identified by the average positions of the surface bridging oxygens. Atomic density maps within the planes parallel to the surface were also computed for selected atoms, namely Lys side-chain nitrogen (Nζ), Glu side-chain oxygens (Oε), C-terminus carbonyl oxygen (O-term.), and N-terminus nitrogen (N-term.), at variable distance from the surface for each simulation. The orientation f
f
of CεNς and CδOε vectors with respect to the surface was defined by the angle with the surface normal b n (with b n coinciding with the z axis). Potential hydrogen bonds were identified when donor-acceptor distances were smaller than 3.3 Å and the angle formed by the hydrogen-donor-acceptor triplet was smaller than 30°. Peptide/surface adsorption free energy was calculated using the probability ratio method.42,43 This technique was already
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Figure 3. Atomic density profiles for water, counterions (a, c, e), and peptide atoms (b, d, f) at the muscovite (a, b), TiO2 (c, d), and HOPG (e, f) surfaces. The position of the surface (see text for the definition) is represented by a vertical black line, which is thicker for the layer in contact with peptide molecules (z > 0). For TiO2, the average plane of the terminal oxygens is also shown (vertical red lines).
tested on similar systems and is described in detail.16 The peptide adsorption free energy, ∆Gads, was calculated as:
∆Gads ≈ -RT
() P
∑ Pi ln P0i ∆di i
(1)
where R is the ideal gas constant, T is the absolute temperature, Pi and P0 represent the probability densities of the peptide molecule being at two distance intervals from the surface, ∆di is the width of the interval (chosen equal to 0.2 Å), and P0 is the normalized probability density of a reference state defined considering positions far away from the surface, where interactions between the interface and the peptide are negligible: P0 was calculated by averaging the Pi value between a defined
cutoff distance for the given molecular system and the maximum distance found. Considering that the cell heights were more than 70 Å for TiO2 and muscovite, distances greater than 35 Å were discarded because the peptides could interact with the image of the bottom of the surface. Thus to define P0 an average value of Pi between 30 and 35 Å was considered as an appropriate choice, whereas in the case of graphite it was calculated for distances in the range 17-20 Å. However, because of the strong interactions between the peptides and the surface it was not possible to sample the full range of distances in each case, and the value of P0 was defined considering the farthest distances from the surface (regions where the peptides were beyond the cutoff distance for nonbonded interactions with the layer). As a
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Figure 4. Angular distributions of the orientation of Lys and Glu side-chain headgroups with the distance from the surface (see text for the definition of the interface).
consequence, larger values of Pi/P0 and thus more negative ∆Gads would have been obtained if the system had been adequately sampled. Thus, the adsorption free energy values obtained from these simulations should be considered as an upper limit. In order to evaluate ∆Gads it was necessary to employ a temperature-accelerated dynamics (TAD) method44,45 in all of the simulations because both peptides remained attached to the surface within the sampled time at T ) 300 K. The TAD technique is used to accelerate the desorption process through simulations at higher temperatures, and then the desorption parameters can be extrapolated at the lower temperature of interest. ∆Gads at 300 K was calculated assuming a linear behavior of ∆Gads between the two (high) temperatures. Even though this is a rough approximation, it may provide a reasonable estimate of ∆Gads. 4. Results and Discussion 4.1. Water Density Distribution and Counterions Location. MuscoWite Mica. As it appears from the examination of K+ density profiles (Figure 2) on both of the muscovite surfaces (the one in contact with water molecules only, that is completely
solvated, and the other one where also the peptides are present, that is partially solvated), K+ ions remain positioned on top of the ditrigonal cavities in the six-member tetrahedral rings of the interface during the whole simulation run at a distance of about 1.5 Å from both the completely and partially solvated (a portion is occupied by the two peptides and thus not reachable by water) surfaces. Their motion on the interface is very limited, and the maximum displacement with respect to the initial position is always lower than 3 Å. These results are in reasonable agreement with earlier investigations,21-23,46-48 indicating that K+ ions have stronger interactions with the negatively charged surface sites than both water hydrogens and positively charged peptide groups. The influence of the adsorbed peptide molecules on the hydration structure in the surface normal direction can be demonstrated through the comparison of water local density profiles for positive and negative values of the z-coordinate shown in Figure 3 (for z < 0, z-, only water molecules are in contact with the surface whereas for z > 0, z+, also peptide molecules must be considered). In the absence of peptides (z-) the mica (0 0 1) surface is covered with a highly structured strongly bound water layer
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Figure 5. Atom-surface distance as a function of the simulation time. Distances of Nζ(Lys) (left-hand side) (a, c, e) and Oε(Glu) (right-hand side) (b, d, f) side-chain atoms from muscovite, TiO2 and HOPG are displayed in (a, b), (c, d), and (e, f), respectively. Peptide 1 color codes: Nζ(Lys1) black, Nζ(Lys3) red; Oε(Glu2) gray and magenta. Peptide 2 color codes: Nζ(Lys1) blu, Nζ(Lys3) dark cyan; Oε(Glu2) olive and green.
where the H2O molecules located in the six-member ring cavities not containing K+ ions, at an average distance of about 3 Å from the surface, are hydrogen bonded to the surface oxygen atoms in the six-member rings and have no direct interactions with each other being more than 5 Å apart. Subsequent layers are identified by the substantially damped oscillations of the density profile extending to about 10 Å above the surface. As it can be noticed observing the region of the plot corresponding to z+, the presence of the peptide causes only a decrease of the local density of water. Evidently notwithstanding the adsorbed peptide molecules, a sufficient number of waters remains close to the surface and only some of the interfacial molecules are replaced by peptide groups. Titanium Dioxide. As far as the TiO2 interface is concerned, the effect of adsorbed peptide molecules on the structure of the interfacial water is very similar to the one already discussed in the case of mica: a decrease of the local density of water. As it can be noticed, the reduction affects just the first water layer that corresponds to molecularly adsorbed water molecules, that is those molecules directly linked through their oxygen atoms to the 5-fold titanium atoms of the surface. The peak position and the shape of the Owat density profile do not change much when the peptide is present (z+). The density profiles for both
exposed surfaces have three distinct peaks at about 2.2, 3.6, and 4.8 Å from the 5-fold titanium plane. These distances correspond to the first layer of molecularly adsorbed waters; to waters interacting with the first hydration layer, terminal and bridging oxygens of the surface; and to waters hydrogen bonded to the second hydration layer, respectively. Ca2+ ions have only one single peak located between the molecularly adsorbed peak and the second hydration peak of water molecules, indicating that Ca2+ ions prefer to bind terminal oxygens and are only partially hydrated. Our MD results predict tetradentate sites for Ca2+, in accord with previous data,49 consisting of two terminal oxygens (including protonated terminal oxygens) and two water molecules at average distance of about 2.3 Å. Ca2+ ions appear to be largely excluded from the first solvation layer but strongly adsorb at the aforementioned sites, remaining there till the end of the simulation. The MD simulation showed an outward shift of about 0.2 Å when the surface is completely hydrated (Figure 2), in reasonable agreement with earlier investigations.49,50 HOPG. As far as HOPG is concerned the equilibrium distance between the water oxygen and the surface is about 3.2 Å in agreement with the previously reported data.51 As observed in
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TABLE 1: Atom-Surface Distances (Å): Statistical Analysis atom
mean
σ
min
max
range
Muscovite 1:Nζ(Lys1) 1:Oε1(Glu2) 1:Oε2(Glu2) 1:Nζ(Lys3) 2:Nζ(Lys1) 2:Oε1(Glu2) 2:Oε2(Glu2) 2:Nζ(Lys3)
9.4 3.0 2.9 3.0 11.9 9.6 9.6 2.4
4.6 0.5 0.4 2.1 2.9 1.8 1.8 0.2
1.6 2.1 2.1 0.8 3.1 4.3 4.8 1.2
17.8 6.2 5.6 12.9 20.1 15.8 15.7 4.5
16.2 4.2 3.5 12.1 17.0 11.5 10.9 3.3
TiO2 1:Nζ(Lys1) 1:Oε1(Glu2) 1:Oε2(Glu2) 1:Nζ(Lys3) 2:Nζ(Lys1) 2:Oε1(Glu2) 2:Oε2(Glu2) 2:Nζ(Lys3)
12.2 2.8 4.1 7.0 3.8 13.1 13.1 3.4
2.3 0.6 0.4 3.1 0.6 1.8 1.7 0.1
5.5 2.2 3.1 3.0 3.1 6.6 6.4 2.9
16.7 6.6 6.6 15.8 8.4 17.1 16.9 4.5
11.2 4.4 3.5 12.8 5.3 10.5 10.5 1.6
HOPG 1:Nζ(Lys1) 1:Oε1(Glu2) 1:Oε2(Glu2) 1:Nζ(Lys3) 2:Nζ(Lys1) 2:Oε1(Glu2) 2:Oε2(Glu2) 2:Nζ(Lys3)
4.7 4.9 4.9 4.3 4.5 6.6 6.7 7.7
0.6 1.0 1.0 0.6 0.5 1.4 1.4 2.1
2.9 3.0 2.7 2.8 2.9 3.6 3.5 2.9
8.0 10.9 10.4 7.2 7.7 12.4 12.7 11.7
5.1 7.9 7.7 4.4 4.8 8.8 9.2 8.8
previous cases, the presence of the peptides causes only a small decrease of the local density of water. 4.2. Peptide Motion, Orientation, Conformational Behavior, and Surface Adsorption Free Energy. In order to understand how the peptide side chains behave in close
proximity of the surface, the orientation dependent density distributions of Lys and Glu side-chain headgroups were examined. Atomic density maps within the planes parallel to the interface were computed for Nζ (Lys amine nitrogen) and Glu carboxyl oxygens at variable distance from the surface using f
f
the angles between the CεNς (Lys) and CδOε (Glu) vectors and the surface normal direction (z axis). The maps are shown in Figure 4. Peptide side-chain dynamics and backbone conformational behavior were characterized by the evolution of atom-surface distances (Figure 5) with the corresponding statistical parameters (Table 1) and Ramachandran maps of the backbone dihedral angles. Titanium Dioxide. The examination of peptide group-based local density profiles (GBLDPs) reveals that the first layer is mainly composed of the carboxylate group (about 2.2 Å from the surface), whereas the Lys charged side chain is accommodated into the second layer (at a distance of about 3.2 Å from the surface) together with the Lys nitrogen atoms which, on the other hand, were also found far away from the surface. The C-terminus group instead is more than 5.2 Å from the interface. At this interface the adsorbed Lys side-chain headgroups (Nζ) are predominantly located at 3.2 Å from the surface and oriented toward the interface with an angle in the 150-180° range. As a consequence, the Nζ hydrogens are involved in hydrogen bonding interactions with the surface oxygens. Glu COO- side chains take instead two different orientations: the carboxyl oxygens closer to the surface (about 2.2 Å) are almost perpendicular to it when adsorbed on Ti sites, whereas those at about 3.7 Å are mainly parallel to the surface (range: 70-100°).
Figure 6. Four frames extracted from the MD simulation of the adsorption of KEK on TiO2. One of the Glu side chains (COO group) detaches from the titanium atom (brown atom) of the surface and binds directly to a Ca2+ ion (orange atom). Peptide atoms are shown in stick mode (hydrogen, white; nitrogen, blue; oxygen, red, carbon gray), whereas surface atoms (titanium brown, oxygen red) and calcium ions (orange) are displayed as balls. Water is omitted for clarity.
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Figure 7. Four frames extracted from the MD simulation of the adsorption of KEK on muscovite. One of the Glu side chains (COO group) remains bound to the surface till the end of the simulation through the interaction with K+ ions (magenta atoms). Peptide atoms are shown in stick mode (hydrogen, white; nitrogen, blue; oxygen, red, carbon gray), whereas surface atoms (aluminum gray, silicon dark cyan, oxygen red, hydrogen white) and potassium ions (magenta) are displayed as balls. Water is omitted for clarity.
Both peptides were stably adsorbed on TiO2 attaching the side-chain groups of both Lys and Glu amino acids to the layer. Indeed, as already demonstrated in earlier works,9-11,14-17 there is a strong affinity between each individual Lys/Glu amino acid and the TiO2 surface, and a wide variety of adsorption modes are observed: hydrogen bonding interactions between the NH3+ groups and the surface oxygens (terminal and/or bridging oxygens including hydroxyl moieties), monodentate and/or bidentate coordination of carbonyl and/or carboxyl groups to the 5-fold titanium sites, hydrogen bonding of the carbonyl and/ or carboxyl moieties to the hydroxyls of the surface. However, during the present simulations another very interesting effect was observed. The carboxyl oxygen was coordinated to a titanium site at the beginning of the simulation, but it was also located in close proximity to a Ca2+ ion (adsorbed between two terminal oxygens). This oxygen detached from its original binding site and reoriented itself toward the Ca2+ ion during the last 4 ns of the simulation. This behavior is clearly visible in Figure 6 and suggests that Ca2+ ions, as well as K+ ions in the case of mica (Figure 7), can compensate for both the carboxyl group and the surface charges. This data appear to support the view that the adsorption of COO- groups can occur via Ca2+ ions acting as a bridge between the peptide and the surface. Calcium ions were found to be practically locked in the interfacial region subsequent to their adsorption. No calcium ions were found in the aqueous bulk, indicating that they constitute an integral part of the interface. Indeed, Ca2+ ions appear to be strongly bound to the interfacial region (Figure 2), thereby inducing a gluing effect on the peptides. As a consequence, the formation of tightly packed surface-peptide complexes occurs. MuscoWite Mica. A deeper inspection of the peptide GBLDPs reveals the nature of the peptide-surface interaction and how
the various groups are responsible for peptide adsorption. The first layer is mainly composed of the charged amino groups of the Lys side chains and the carboxylate groups of the Glu side chains whereas the C- and N-terminus nitrogens together with the backbone nitrogen of Lys residues are inserted into the second water layer. The NH3+ moieties prefer positions above the surface cavities surrounded by two Al atoms with respect to positions in cavities surrounded by one or zero Al atoms, in agreement with data reported in the literature.52 The peptide molecules are mainly adsorbed on the surface through hydrogen bonding interactions between the positively charged amine groups and the surface oxygen sites and by means of K+mediated indirect interactions of their negatively charged carboxylate groups. No apparent lateral or vertical displacement of the adsorbed peptides was observed during the whole simulation run (more than 20 ns), indicating that the peptides are captured by the surface and have strong interactions with specific sites (mentioned above) of the layer. At the mica interface the side-chain headgroup behavior is quite different from the TiO2 case but very similar to the HOPG one (see next paragraph) even though in the latter case (that is HOPG) the distributions are very broad and explore different angular regions of the map because of the absence of specific localized interactions. The higher Nζ peak corresponds to sidechain headgroups at a distance of about 2.4 Å from the surface forming an angle of about 85° with the surface normal, and the hydrogen atoms point toward the layer engaging hydrogen bonds with the interfacial oxygens. Comparison of this distribution with the TiO2 one reveals that Nζ headgroups are less inclined with respect to the surface plane (in the case of TiO2 the average b angle is about 165°). Glu side chains orient their C δOε vectors
Adsorption of Ionic Peptides on Inorganic Supports in the range 40-80° with a maximum at about 60°. It can be noticed that the distribution peaks of both headgroups (both Lys and Glu side chains) are sharper for TiO2 than for mica, suggesting that these moieties are less mobile on TiO2 at least during the 20-ns time of the simulation. The data show that both peptides remain bonded to the surface through their Lys side chains, which form H-bonds with the layer oxygens till the end of the simulation. Peptide 1 is more strongly attached to the interface because of the fact that both Lys1 and Glu2 side chains have contacts with the surface from the beginning of the simulation; moreover, after 21 ns also the flexible Lys3 side chain stretches toward the interface anchoring that part of the molecule to the mica layer. As far as peptide 2 is concerned, only Lys3 is bound to the mica layer, whereas the side chain of the other residues can move more freely and extend into the bulk region of the solvent; however, H-bonds with the nearby peptide are often formed. Indeed, these contacts are very frequent, constantly breaking and forming again during the whole simulation run, and contribute to stabilize the peptide upon the surface. The peptides can interact with each other through their side chains located far from the surface thereby increasing molecular packing. Moreover, visual inspection of the trajectory and a more detailed analysis of the interfacial region have allowed us to identify a possible role played by the potassium charge balancing counterions during the adsorption process. Indeed it was observed that the negatively charged carboxyl groups have preferential interactions with the K+ ions and through them anchor quite stably to the interface (Figure 7). HOPG. The density distributions of the peptide side-chain groups (Figure 4) have several wide peaks that suggest that these moieties can be located at different distances from the surface and can adopt various orientations with respect to the layer normal because of the absence of specific localized interactions. However, as evidenced by the two peaks at about 3 Å visible in the GBLDPs (Figure 3) both the amine N- and amide C-terminus groups can be found quite near the surface because that upon binding, the protons of these groups are attracted by the quadrupole moment of the carbon rings. Notwithstanding this, no preferential adsorption site for the peptides is identified and all peptides frequently change their location, orientation, and conformation upon the layer. The results are consistent with the experimental observations that soft biomolecules adsorb on hydrophobic surfaces undergoing large conformational changes.53,54 In general it can be said that the molecules are weakly bonded to the interface, but the dipolar nature of the different portions of the molecules promotes their adsorption. In accord with the study of Roman and co-workers,55 a favorable adsorption pathway is due to the presence of deprotonated carboxyl and fully protonated amine groups, but the binding is weak as confirmed by the high mobility of the molecules on the surface. As far as peptide backbone conformational characteristics are concerned, it has been observed that for all model surfaces, that is muscovite, TiO2, and HOPG, peptide backbones are not conformationally rigid but slowly evolve in their conformational space folding into different secondary structures. The Ramachandran maps of each single residue (not shown, see ref 18) confirm this view. All the folded regions of the maps, that is β-sheet, right-handed R-helix (RR), and left-handed R-helix (RL), were explored, and the data resembles the results obtained in pure solution,18 suggesting that the adsorption of randomly assembled unfolded peptides on mica and TiO2 is primarily realized through side-chain interactions and as a consequence
J. Phys. Chem. C, Vol. 113, No. 6, 2009 2441 the interface does not necessarily represent a limit or a constraint to the conformational characteristics of the backbone atoms of the molecule. However, as already observed in a previous investigation,17 supramolecular folded structures are able to preserve the arrangement they adopted in solution. Surface Adsorption Free Energy. Because of the presence of distinct interaction sites, which link the various groups of the peptides in different ways and thus confine the molecules to specific areas of the surface, it was interesting to evaluate and compare the adsorption free energy in the case of mica and TiO2. In both models, as the peptides remained attached to the surface within the total simulation time at 300 K, it was necessary to employ the TAD technique44,45 in order to evaluate ∆Gads. MD simulations of peptide/mica desorption were carried out at 600 and 900 K (total simulation time ) 13 ns), whereas for TiO2 they were performed at 800 and 900 K for a total simulation time of 10 ns. From the sampled data the average ∆Gads was extrapolated at T ) 300 K via an Arrhenius plot, and it was roughly -7.6 ( 1.1 and -10.9 ( 1.9 kcal/mol for mica and TiO2, respectively. As shown from the reported data, the peptide adsorbs on both inorganic layers, and its adsorption is the result of several contributions from different types of nonbonded interactions involving mainly the side chains. In TiO2 a greater number of multiple coordinations together with hydrogen bonding determine the formation of a stronger peptide-surface complex, as it appeared from the comparison of the adsorption free energies of the two models at T ) 300 K and at T ) 900 K, where the values were found to be about -7.4 and -6.8 kcal/mol for TiO2 and mica, respectively. 5. Conclusions Classical MD simulations have provided some insights into the conformational dynamics of small ionic peptides when located near the rutile (110), muscovite (001), and HOPG surfaces. The stability of the adsorbed configurations has provided further evidence that, in agreement with experimental data, small peptide molecules containing alternating positively and negatively charged residues and their aggregates can “weakly” adsorb on HOPG and “strongly” adsorb on both titanium dioxide and muscovite, engaging direct and indirect interactions with the surface atoms through their amino acid side chains. After their adsorption, calcium and potassium ions were found almost locked in the interfacial region, located on top of TiO2 and mica surfaces, respectively, and were actively involved in peptide/surface adsorption acting as a bridge between the COO- groups of the peptide and the inorganic layers. The anchoring points, identified in the case of TiO2 and muscovite, were strong enough to hold the peptides close to the surface for the whole simulation time and also prevented them to diffuse over the surface. On the contrary, detailed analysis of the HOPG trajectory indicated that while some attractive interaction occurred among the side chains and the π density of the interface, it was not strong enough to keep the peptides tethered to the surface, with the water molecules being successfully able to out-compete the peptide functional groups for these same interactions and, as a consequence, the overall behavior was essentially a random translational motion of the peptides over the surface. Acknowledgment. We thank Andrey G. Kalinichev for providing coordinates and parameters of muscovite (001). Most of the calculations reported in this paper were carried out at the CINECA Supercomputing Center (Bologna, Italy).
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