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Accelerated molecular dynamics study of the effects of surface hydrophilicity on protein adsorption Christian Mücksch, and Herbert M. Urbassek Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b02229 • Publication Date (Web): 17 Aug 2016 Downloaded from http://pubs.acs.org on August 21, 2016

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Accelerated molecular dynamics study of the effects of surface hydrophilicity on protein adsorption Christian Mücksch and Herbert M. Urbassek∗ Fachbereich Physik und Forschungszentrum OPTIMAS, University of Kaiserslautern, Erwin-Schrödinger-Straße, D-67663 Kaiserslautern, Germany E-mail: [email protected]

Abstract The adsorption of streptavidin is studied on two surfaces – graphite and titanium dioxide – using accelerated molecular dynamics. Adsorption on graphite leads to strong conformational changes while the protein spreads out over the surface. Interestingly, also the adsorption on the highly hydrophilic rutile surface induces a considerable spreading of the protein. We pin down the cause for this unfolding to the interaction of the protein with the ordered water layers above the rutile surface. For special orientations, the protein penetrates the ordered water layers and comes into direct contact with the surface where the positively charged amino acids settle in places adjacent to the negatively charged top surface atom layer of rutile. We conclude that for both surface materials studied, streptavidin changes its conformation so strongly that it loses its potential for binding biotin. Our results are in good qualitative agreement with available experimental studies. ∗

To whom correspondence should be addressed

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Introduction Due to the complex 3-dimensional geometry of proteins and the various possible ways of interacting with surfaces protein adsorption forms a quite active field of research 1,2 with more than 3,000 publications each year according to Web of Science and a wide range of medical implications such as biomaterials 3 or therapeutic proteins 4 to name a few. Over the years there have been many in vitro studies dealing with various aspects of the nonspecific adsorption process employing methods such as atomic force microscopy (AFM) 5–9 and scanning force spectroscopy (SFS), 10,11 circular dichroism (CD), 12–14 Fourier transform infrared spectroscopy (FTIR), 15,16 differential scanning calorimetry (DSC) 12,15,17 and many more. A direct way of looking at the underlying atomistic mechanisms of protein adsorption are in silico studies with molecular dynamics (MD) being among the most popular ones. 18–26 One of the major aspects when studying the interactions between proteins and surfaces are specific characteristics such as wetting properties, topography and crystal orientation of the surface. In this study we want to compare two widely different surfaces, namely mildly hydrophilic graphite in its pure state 26 and a hydrophilic titanium dioxide surface in the form of rutile which represents the surface oxide layer of pure titanium. 27 Both surfaces are used as implant material 28–30 due to their biocompatibility and have a broad history of computational adsorption studies. 23,31–34 We note that the effects of the surface hydropathy on protein adsorption is an active research field. 2 As a model protein for our adsorption studies we have chosen streptavidin which is known for its binding with the vitamin biotin that is considered as one the strongest non-covalent interactions in nature. This feature made it a most interesting target for one of the earliest biological steered MD simulations. 35 The native form of the protein consists of a homotetramer which can bind up to four biotin molecules. 36 Furthermore, streptavidin can be coupled to a solid support while biotin is coupled to a moiety of interest making this a widely used application for assays. 37,38 Therefore, it is of significant importance to know if the streptavidin structure is affected by the surface. 2


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The new aspect in this study is the use of advanced sampling methods, namely accelerated MD, 39 to achieve a more realistic comparison of protein adsorption on two different surfaces than previous studies 32 since these processes demand long simulation times 24 in order to obtain useful predictions about the final structure. In order to better compare the fundamental processes on these surfaces only perfect and clean surfaces were considered with disregard to any secondary effects that depend on various conditions.

Methods

Figure 1: Composition showing streptavidin in an end-on orientation (orientation 1) above the (left) graphite (0001) (right) rutile(110) surface in 0.1 M NaCl. Nonpolar residues are shown in blue, neutral residues, meaning uncharged and not very polar residues in green, and polar residues in red. The classification of amino acids is based on sidechain hydrophobicity measurements at pH 7 by Monera et al. 40 which is close to the pH of our structural data. Water molecules are not shown for clarity while sodium ions are displayed in yellow and the chloride ions in cyan. The graphite surface is shown in grey, while the rutile surface is colored red (O) and pink (Ti). The MD simulations were carried out with NAMD 2.9 41 in combination with the CHARMM27 force field 42 and the TIP3P water model 43 for the explicit solvent. To ensure a 2 fs time step the SHAKE algorithm 44 was used. The long-range electrostatics were handled by the particle-mesh-Ewald (PME) routine 45 in periodic boundary conditions. A temperature of 298 K and a pressure of 1 atm were ensured for all simulations. The structural data of streptavidin was taken from the protein data bank with PDB3


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ID:1STP. 36 Since each of the monomeric units with residues 13 through 133 as the core protein has a high affinity for biotin 36 and due to computational constraints only the monomer structure is studied here. We note that it was observed that the streptavidin tetramer appears to collapse upon adsorption on graphite. 46 We excluded the biotin molecule from our simulation since we wanted to study how the streptavidin molecule itself adsorbs and if, after adsorption, a biotin interaction might be possible or not. The protein structure at a pH of 7.8 and a resulting net charge of +2 e is neutralized in a 0.1 M NaCl solution. An image composition showing the protein above the two surfaces can be seen in Fig. 1. The protein orientation in this figure, which we term orientation 1, is in an end-on orientation. Orientation 2, which is obtained from orientation 1 by a 90◦ rotation around an axis parallel to the surface is in a side-on orientation. Both orientations initially have a minimum distance of 6 ˚ A to the surfaces and are compared in Fig. 2. For this study we considered the (0001) surface of graphite and the (110) surface of rutile. While graphite has a perfectly flat topography, the rutile surface shows a corrugation on the atomistic level with the O atoms sticking above the Ti atoms; due the negative charge on the bridging oxygen atoms and positive charge on the Ti atoms the (110) surface is polar. 47 We consider only perfect surfaces without surface defects and incorporation of contamination effects. On titanium dioxide, titanium atoms may be hydroxylated and bridging oxygens may be protonated to a certain degree depending on the experimental conditions 48,49 but for simplicity and better comparison with previous work 32 these effects are disregarded here resulting in clean stoichiometric surfaces. So parameters for describing the interactions with clean graphite were the same as in our previous work 26 as described by Wu and Aluru. 50 The resulting macroscopic contact angle on the graphite surface amounts to 55◦ . 26 Note that this procedure for describing a clean and flat surface is in agreement with the experimental procedure applied by Cooper et al. 46 The rutile surface was set up with lattice parameters of a = 2.95812 ˚ A and c = 4.59373 ˚ A and comprised of 3 TiO2 layers; this thickness is considered as adequate by Bandura and Kubicki. 51 The charges and Lennard-Jones parameters

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were taken from Utesch et al. 32 On this surface, we observed complete spreading of simulated water droplets, i.e., a contact angle of 0◦ . Both surfaces had lateral sizes of 80 × 80 ˚ A2 to allow for sufficient movement of the protein; during the simulations the surface atoms were fixed. To characterize the surface properties short simulations of 6 ns using classical MD in pure water boxes were carried out. This allowed us to obtain density profiles 52 of water near the surfaces, see Fig. 3 below. To stabilize the crystallographic structure of streptavidin and the explicit water molecules inside the simulation box energy minimization and equilibration simulations were carried out for 200 ps. The following adsorption simulations were performed using the implementation of dual accelerated MD 39,53,54 into NAMD. 55 In order to better compare the simulation outcome all configurations used the same dihedral acceleration and the total acceleration was scaled according to the system sizes as described in our previous work. 56 A total of 2 · 107 time steps was used to achieve an adequate sampling of the adsorption process. Note that a ‘real time’ cannot easily be estimated due to the bias in the dynamics, especially for complex systems such as the proteins considered here. 57 Unlike our previous work 58 there is no experimental adsorption kinetics data or independent theoretical information of the transition states available that could help in relating the time spent in accelerated dynamics to the real time. Also, we are more interested in the final adsorption state than the dynamics of the adsorption process.

Results and Discussion Let us first take a look at the surface-water interactions of the hydrophilic rutile surface and the more hydrophobic and non-charged graphite surface. Fig. 3 shows in the right-handside the water density profile – more precisely: of O atoms in water – above the different surfaces averaged over the last 100 ps of the MD trajectories in pure water. Above the rutile

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Figure 2: (Top) Streptavidin orientations shown above the graphite(0001) surface. Color coding is equivalent to Fig. 1. Orientation 1 is in an end-on orientation, orientation 2 in a side-on orientation. These starting orientations are used identically for the titanium dioxide simulation. (Bottom) Partial charge distribution on the protein surface.

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surface, we observe the water layer structure proposed in 32,48,49 with significant maxima in the density profile at 1.4 ˚ A and 2.45 ˚ A; which can be attributed to the first water layer as also seen in the snapshots of Fig. 3. A strong ordering of the water molecules in the first layer can be observed. The second peak in the density profile for water on rutile can be attributed to water molecules with their O–H bonds aligned almost perpendicular to the surface so that here hydrogen bonds to the surface oxygens form. Quantitatively the orientation of the water O–H bonds can be characterized by their angle ϑ towards the (outward) surface normal. The O–H bonds arising from the second peak in the density profile point towards the surface, with a mean angle of ϑ = (159.9 ± 10.5)◦ . The other O–H bonds arising from water molecules situated between the bridging oxygens and responsible for the first peak in the density profile point parallel to the surface, ϑ = (92.5 ± 18.8)◦ . The first density peak corresponds to water molecules which are positioned in between the bridging oxygen atoms of the surface forming hydrogen bonds as well. A second water layer with clear ordering above rutile is seen as two more peaks in the density profile at 4 ˚ A and 5.05 ˚ A. The orientation of the water molecules in this layer is based upon the first layer so that the hydrogen bonding network is optimized. In contrast, above the graphite surface a depletion of water molecules can be observed as expected for a more hydrophobic surface where no hydrogen bonds can form. Almost the whole first water layer as observed on rutile would fit in between the empty space on graphite. The water layers on graphite also show a quite different H–bond pattern where the favored direction is horizontal instead of the vertically oriented hydrogen bonds observed on rutile. In a further simulation we studied the stability of the streptavidin monomer in a water environment. To this end we performed an accelerated MD simulation for the same simulation time of 2 · 107 time steps, in which the protein is contained in the water box with ions but without any surfaces. The resulting evolution of the root mean square deviation of streptavidin is shown in Fig. 4. We conclude that the protein structure remains stable in

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Figure 3: Water layers on graphite and rutile obtained from simulations in pure water. The density profile of oxygen atoms of the TIP3P water molecules is shown in the right-hand side. The first two peaks in the density profile of rutile belong to the first water layer whereas the next two peaks belong to the second water layer. The coloring of the layers in the snapshots is motivated by the peaks in the density profile. Furthermore, hydrogen bonds are shown as red dashed lines in the snapshots. In water, small spheres represent H, and large spheres O. solution for the time scale of our simulations; this result is corroborated by the absence of any significant changes in the secondary structure, see Table 1. The presence of a surface strongly affects the protein structure. Here, the graphite surface has a stronger impact on the protein conformation than rutile as expected for a more hydrophobic surface. We note that the protein closely approaches the surface. For graphite the minimum distance of a protein atom to the surface averaged over the last 2 ns amounts to 2.31 ± 0.09 ˚ A for both orientations. For rutile the distance is closer, only 1.24 ± 0.08 ˚ A for the favorable conformation 2, but it stays rather distant to the surface, 4.02 ± 0.91 ˚ A for orientation 1; the origin of this difference will be discussed below. The protein-surface distances show similar features as the distance of the first water layer to the surface given in Fig. 3. Therefore we argue, that no water molecules can be present between the adsorbed protein and the surface. This dehydration of the surface is well-known for hydrophobic surfaces, but here also occurs for rutile. A clear difference in the adsorption behavior between graphite and rutile can be seen 8


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45 No Surface

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Figure 4: Root mean square deviation of the streptavidin monomer during adsorption and in pure water (without a surface) for comparison. Acceleration was applied to all simulations.

Figure 5: Adsorption snapshots after 2 · 107 time steps at the exact same scale.

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in the amount of spreading on the surface; this is shown by the adsorption snapshots in Fig. 5 and demonstrated quantitatively by the protein’s radius of gyration component parallel to the surface in Fig. 6. The protein’s tertiary structure on graphite is almost completely destroyed. Also, its secondary structure content is greatly reduced as can be read off Table 1. The adsorption process on graphite follows the usual pattern as described in previous work. 23,56 The protein almost completely unfolds and spreads out on the surface guided by the attractive van-der-Waals interactions shown by Fig. 7. This is in perfect agreement with experimental findings of streptavidin on graphite by Cooper et al. 46 Orientation 1, which is starting in an end-on orientation, has less surface contact in the beginning and so more flexibility for spreading during the adsorption process on graphite. 45 Graphite Orientation 1

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Figure 6: Radius of gyration of the streptavidin monomer (component parallel to the graphite surface). In contrast to the results for graphite, the adsorption behavior of streptavidin on rutile may come as a surprise. Interestingly, this hydrophilic surface is also able to greatly disturb the protein structure. These structural alterations can be detected in the root mean square deviation (Fig. 4) and in the protein’s radius of gyration (Fig. 6). But when looking at the adsorption snapshots on rutile (Fig. 5) one can clearly recognize that a great deal of secondary structure was destroyed; see Table 1 for a quantitative comparison. However, the

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Figure 7: Van-der-Waals (vdW) and electrostatic (elec) interaction energies between streptavidin and surface atoms, i.e., graphite and rutile. The blue curve for the van-der-Waals interactions for orientation 1 on rutile is hidden behind the van-der-Waals interactions for orientation 2 since it is almost zero. streptavidin monomer has not spread out on rutile as strongly as on graphite and rather kept parts of tertiary structure. Fig. 7 demonstrates a strong influence of the starting orientation on the adsorption pathway. In contrast to graphite, adsorption on rutile is guided via electrostatic interactions, where the initial adsorption phase takes a much longer time than on graphite. It becomes apparent that the first two water layers which are the most significant layers also play an essential role in the adsorption process. Based on the observation from Fig. 7 we see that the starting orientation 2 develops very strong attractive electrostatic interactions with the titanium dioxide surface. By further analyzing individual energetic contributions the positively charged amino acids ARG53, ARG59, ARG84 and LYS80 can be identified as the key components for this strong electrostatic interaction in the case of orientation 2. This is plausible since the top layer of the (110) rutile surface consists of negatively charged O atoms. Our analysis for starting orientation 2 shows that the streptavidin molecule is able to permanently penetrate the first water layer. This direct contact with the rutile surface mainly established via these above mentioned

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positively charged key amino acids keeps the protein in a locked position throughout the advancing adsorption process. In Fig. 8 we display the electrostatic interactions between the protein and the first two water layers; to this end, all water molecules in a distance of 0–2.9 (2.55–5.5) ˚ A from the surface – based on the extension of the water layers shown in Fig. 3 – are taken to make up the first (second) water layer. We see that especially the attractive interactions of orientation 2 with the second water layer are stronger than with the titanium dioxide surface itself. These interactions with the water layers and the titanium dioxide surface give an explanation for the strong unfolding of the protein (see also Table 1) which is optimizing its structure according to the charge distribution. The water layers are also key for explaining the partly unfolded structure of starting orientation 1 on rutile. This orientation has little or none direct interactions with the titanium dioxide surface as shown in Fig. 7. It rather adsorbs via the second water layer which has more attractive interactions with the protein than the first layer (see Fig. 8). The fact that adsorption on titanium dioxide surfaces can mainly be attributed to the first two water layers is in agreement with previous simulational results. 31,32 The improved simulation time scale due to the acceleration is already enough to greatly disturb the protein’s tertiary and secondary structure during the adsorption of orientation 1 via the water layer structures. It is mainly the altering attractive and repulsive interactions with the water layers (Fig. 8) and surface (Fig. 7) that are responsible for tearing the protein structure apart. Possibly, though, a longer simulation time would result in an even further unfolding also for this orientation since the adsorption on the second water layer for orientation 1 occurred only in the last third of the adsorption phase and a penetration of the first water layer as seen for orientation 2 might be possible when continuing the adsorption process longer. The charge distribution in the protein displayed in Fig. 2 suggests that indeed orientation 1 probably has a less favorable starting position since many negatively charged protein parts are pointing towards the surface. This conclusion is corroborated by our finding

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that the protein in orientation 1 approaches the surface less closely than in orientation 2, see our discussion above. In all adsorption cases studied here, whether on graphite or rutile, the biotin binding site that was located in the protein interior 36 is now completely destroyed; its components are now found at the protein exterior rendering the adsorbed streptavidin monomer unable to bind biotin anymore. Quantifying this, we found high RMSD values of 32.71 ˚ A (graphite orientation 1), 20.82 ˚ A (graphite orientation 2), 17.53 ˚ A (rutile orientation 1) and 19.82 ˚ A (rutile orientation 2) for the biotin binding site. first water layer Orientation 1

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Figure 8: Electrostatic interactions on the rutile surface between streptavidin and the first two water layers, averaged over an interval of 2 · 104 time steps. The assignment of the water molecules to the two water layers described in Fig. 3 was dynamically updated. Table 1: Secondary structure calculated using DSSP 59 of the initial streptavidin monomer and after 2 · 107 time steps of accelerated MD for the various starting orientations (‘Or’) and the simulation case in absence of any surface. after 2 · 107 time steps

initial minimized structure α-helix 310 -helix β-sheet

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Conclusions In this work we performed accelerated MD simulations to study adsorption processes of the streptavidin monomer on graphite and rutile surfaces on a more realistic time scale. Advanced sampling is needed to overcome metastable states occurring while the protein is optimizing its configuration on the surface. 56 The two clean and stoichiometric surfaces show clearly different wetting properties: while our graphite surface displays a mildly hydrophilic character as previously shown, 26,60,61 the titanium dioxide surface is strongly hydrophilic inducing formation of a multiple water-layer structure. 32,48,49 Depending on the surface and the initial orientation of the protein different adsorption mechanisms come into play. On graphite the protein optimizes its structure by spreading out due to entropic reasons. 2 This spreading and unfolding of a peptide agrees with experimental observations 7–9 and has in particular been observed for the protein and surface studied in the present work. 46 For our graphite model only van-der-Waals interactions with the protein are possible so orientations that start with the least surface contact have the highest flexibility for spreading, 62 as seen for our end-on orientation of streptavidin. In comparison with adsorption on titanium dioxide this process on graphite results in a higher unfolding degree which is also happening faster. On titanium dioxide electrostatic interactions with the surface and the induced water layers play a key role for the adsorption process. The protein can either adsorb via the water layers – especially the second water layer can be attractive – or it can even penetrate the first water layer to come into direct contact with the rutile surface. Due to the nature of electrostatic interactions originating from the surface and/or the water molecules inside the water layers, which can either be attractive or repulsive, the protein structure can greatly be disturbed while adjusting to the charge distribution. The starting orientation also plays a role, since orientations which put protein charges of the same polarity close to the surface are less favorable to adsorption. Our findings of an unfolding and spreading protein on a hydrophilic titanium dioxide surface are in good qualitative agreement with studies conducted 14


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by Raffaini and Ganazzoli. 34 These authors used other proteins in their study; in addition, their simulations do not employ explicit water and hence the ordered water layers above the TiO2 surface are absent. However, their study can be compared to our scenario with starting orientation 2 where direct contact with the rutile surface has been established. Due to the high unfolding degree and the loss of the biotin binding site of streptavidin on both surfaces we argue that biotin binding after adsorption of the protein is unlikely. Also the attachment of biomolecules via the strong streptavidin-biotin bond onto these surfaces with pre-adsorbed streptavidin might be questionable. Therefore, it is recommended that nonspecific adsorption of any protein should be avoided if surface functionalization is desired. 63 It remains unclear whether the native tetrameric form of streptavidin would have been able to withstand unfolding on the surfaces or whether an already bound biotin molecule might have been able to alter the adsorption outcome. These questions may be answered by further investigations. We note that the relevance of our results for medical applications of titanium implants is not straightforward. The well-known biocompatibility of Ti does not only originate from the ease of adsorption of various proteins on the surface or from their non-denaturalization during adsorption. Also the subsequent protein-cell interactions are decisive to determine the cellular response. Furthermore, the bacterial protein studied here which shows a high unfolding tendency is not of importance considering the biocompatibility of medical implant surfaces. One limitation of our present study is the use of perfect and clean surfaces, whereas in experiment the surfaces will as a rule be contaminated and contain defects. While it is possible to incorporate effects like hydrocarbon adsorption 26,60,61 on both surfaces or hydroxylation and protonation 48,49 on titanium dioxide into our model, it is not a priori clear which of these factors should actually be modeled. Depending on the experimental treatment like photoinduced hydrophilicity by means of UV irradiation on titanium dioxide 49 various surface modifications are possible.

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Acknowledgement We acknowledge financial support by the Deutsche Forschungsgemeinschaft within project Ur 32/26-1. Furthermore we appreciate the computational resources provided by the compute cluster ‘Elwetritsch’ of the University of Kaiserslautern.

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