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J. Phys. Chem. B 2004, 108, 13850-13854
Surface Ordering of Proteins Adsorbed on Graphite Giuseppina Raffaini and Fabio Ganazzoli* Dipartimento di Chimica, Materiali e Ingegneria Chimica “G. Natta”, Sez. Chimica, Politecnico di Milano, Via L. Mancinelli 7, 20131 Milano, Italy ReceiVed: May 25, 2004; In Final Form: July 2, 2004
The surface-induced rearrangement of distant protein strands adsorbed on a hydrophobic graphite surface is investigated through atomistic molecular dynamics simulations and energy minimizations. We show that fragments both of globular proteins consisting of either R-helices or β-sheets and of a fibrous protein containing a triple helix do form parallel strands on this surface, irrespective of their native structure. On the other hand, a new secondary structure consisting of β-sheets lying on the surface was never observed even after a long simulation time. The parallel ordering is characteristic of graphite being absent, for instance, on a hydrophilic poly(vinyl alcohol) surface. Our simulations indicate that this result is not related to the surface rigidity, and we suggest that it is due to a combination of the surface hydrophobicity, crystallinity, and smoothness.
Introduction Protein adsorption on heterogeneous surfaces is important in many practical cases, such as a biomaterial implant in a living body, the cell growth in a culture, or the functionality of a biosensor.1 It is also of a nontrivial theoretical interest, given the difficulty of describing the interaction of a very specific amphiphilic copolymer with a surface.2,3 This phenomenon is not easily amenable to a detailed theoretical analysis because of proteins’ specificity, apart from very general features. Therefore, in recent years molecular simulation methods have offered many important insights. In particular, Monte Carlo (MC) and molecular dynamics (MD) techniques have been widely used.4-8 Monte Carlo methods have the great advantage of providing an efficient sampling of the configurational phase space, although for practical reasons they are often restricted to coarse-grained models. Such models obviously neglect the real stereochemical features of proteins, and therefore they can only yield very general results. In particular, all local details are ignored, including also in general the secondary structure (i.e., R-helices and β-sheets). Additionally, the computational efficiency of MC methods is often enhanced by the further simplification of adopting a lattice model, which in principle may severely constrain or affect the results. Conversely, MD techniques do follow the time evolution of the system and can be carried out at a constant (average) temperature. Moreover, atomistic models in continuous space are typically employed in MD simulations, thus yielding in principle realistic predictions about both the kinetics and the thermodynamics of the simulated process. On the other hand, the MD methods are computationally much heavier, and therefore a reliable sampling of the configurational phase space can be sometimes difficult to achieve, so that conformational changes at very long times might be missed. One interesting feature of protein adsorption on flat surfaces recently obtained through coarse-grained MC simulations on a lattice consists of their denaturation and eventually rearrangement, or “refolding”, to a different ordered arrangement.5,6 For instance, a bundle of R-helices was found5 to refold to a “surface * To whom correspondence
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native state” with long parallel strands that were described as a β-sheet structure. The adopted model consisted of an amphiphilic copolymer which did account for the chain stiffness favoring the formation of parallel strands on the lattice through an energy penalty for the formation of kinks in one subset of segments but did not allow for hydrogen bonds. These simulations showed that new refolded, or ordered, arrangements may form on a flat continuous surface, even though we stress that the fold details and the formation of a new secondary structure could be directed by the chosen sequence of units6 and by the underlying lattice. We recently started a systematic study of protein adsorption on heterogeneous surfaces through atomistic MD simulations and energy minimizations of globular protein fragments on a realistic graphite surface.7,8 In the present paper, we report our results about the surface-induced ordered arrangement of the adsorbed strands of real protein fragments with different native structures. In particular, we consider the same globular fragments already studied by us7,8 having an unlike secondary structure, namely, two albumin fragments each formed by three R-helices and a fibronectin module containing three β-sheets, together with short random sequences. Additionally, we also present similar results for a fibrous collagen-like protein fragment formed by a triple helix. We show that in all cases the graphite surface induces some parallel ordering of the strands, although no β-sheets do ever appear within the accessible simulation time. After a short summary of the simulation method, we present our main results, and finally we briefly comment about possible limitations of the procedure. Simulation Method All simulations were performed with the InsightII/Discover 2000 package, distributed by Accelrys Inc.9 (San Diego, CA), using the consistent valence force field10 CVFF with a Morse potential for the bonded atoms. This force field describes nonbonded interactions through van der Waals and Coulombic terms only, with no extra terms for the hydrogen bonds, and like most force fields it does not account for the molecular or surface polarizability. The graphite planes were prepared as described in ref 7, while the initial coordinates of the nonhydrogen atoms were obtained from the Protein Data Bank:11
10.1021/jp0477452 CCC: $27.50 © 2004 American Chemical Society Published on Web 08/14/2004
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human serum albumin, 1AO6; fibronectin type I module, 1FBR; and a collagen-like fragment, 1BBE. Details on the simulation procedure can be found in refs 7 and 8. However, in our first exploratory paper,7 we considered all the side groups of the albumin fragments in their neutral, uncharged form, with only the terminal groups in a zwitterionic form. In the present paper, we considered again the side groups of the albumin fragments in the uncharged form, but we checked also for possible artifacts by taking also into account the appropriately charged side groups at pH ) 7, as already done for the fibronectin module.8 No major effect was detected in practice (see below). The protein fragments were initially placed close to the surface and simply optimized in an effective dielectric medium with a distance-dependent dielectric constant corresponding to water ( ) 78). The MD simulations were then performed at a constant average temperature (T ) 300 K) controlled through the Berendsen thermostat.12 The dynamic equations were integrated with the Verlet algorithm using a time step of 1 fs. In all cases, the runs lasted for at least 1 ns. After an initial, possibly stepwise decrease, the total and potential energy eventually fluctuated around a constant value, indicating achievement of the equilibrium state. The most stable adsorbed geometries were then obtained after optimization of a large number of instantaneous snapshots. Additional MD runs in water of these systems were used to analyze the stability of the adsorbed geometries. These simulations were carried out by adding a few thousands of water molecules to a cell much larger than the whole system with periodic boundary conditions, adjusting the water density to 1 g cm-3, and setting ) 1, while the runs lasted for 10 ps after the initial equilibration.7,8 Results and Discussion protocol,7
According to our simulation we first optimized the geometries of the protein fragments close to the flat graphite surface in different orientations by simple energy minimizations. In the two albumin fragments, each containing three R-helices and short random sequences, we found minor rearrangements with a local loss of the secondary structure for the strands in contact with the surface, independently of the presence of neutral, or of appropriately charged side groups. Such rearrangements were smaller for the fibronectin module (only charged side groups were considered, in this case), and are still smaller for the collagen-like fragment. The interaction energy Eint of all the fragments in the local energy minima corresponding to different starting orientations are plotted in Figure 1 as a function of the number of amino acids in contact with the surface n5Å, where we take 5 Å as the upper limit for a significant contact distance.7,8 Such interaction energy is taken as positive, being defined as the energy required to desorb the fragment and bring it back to the free, native geometry. Overall, we find a linear correlation between Eint and n5Å, the best-fit line through the origin (solid line in Figure 1) being given by
Eint ) 57(3)‚n5Å kJ mol-1
(1)
the value in parentheses being the estimated standard error on the last significant digit. Despite the general behavior, a more careful analysis shows that we can distinguish some differences among the individual proteins, so that the albumin fragments, the fibronectin module, and the collagen-like fragment display slopes equal to 71(3), 54(1), and 41(1) kJ mol-1, respectively. The interaction energy of the albumin fragments is not significantly affected by the presence of the charged side groups instead of the neutral ones, the difference being comprised to within (2%.
Figure 1. The interaction energy, Eint (filled symbols), and the strain energy, Estrain (open symbols), of the protein fragments adsorbed on graphite plotted as a function of the number of residues in contact with the surface n5Å (see text). Each data point corresponds to a local energy minimum found in the initial adsorption stage considering the fragments in different orientations. Upper triangles (2, 4) refer to the two albumin fragments,7 lower triangles (1, 3) to the fibronectin module,8 and circles (b, O) to the collagen-like protein fragment. The solid lines are the best-fit lines through the origin given by eqs 1 and 2.
The surface-induced local rearrangements give also rise to an intramolecular strain energy Estrain, also reported in Figure 1. This energy is obtained as the difference between the values of the frozen adsorbed fragment isolated from the surface and of the free native fragment in the optimized geometry. Again, there is a linear correlation between Estrain and n5Å, the best-fit line through the origin being
Estrain ) 13(1)‚n5Å kJ mol-1
(2)
Small differences among the different proteins are again present, since the albumin fragments, the fibronectin module, and the collagen-like fragment show a slope equal to 17(1), 13(1), and 6.7(7) kJ mol-1, respectively. We stress that in these energy minima found for the initial adsorption in different orientations, both Eint and Estrain somewhat depend on the native structure of the fragments, but the overall similarity is eventually related to the presence of the same natural amino acids and to cooperative effects which blur the specificity of the amino acid sequences, in particular, their hydropathy pattern. However, the main issue borne out by Figure 1 and eqs 1 and 2 consists of the much faster increase of Eint with n5Å compared to Estrain. This feature implies that the protein fragments can undergo much larger deformations to maximize their surface interaction. Accordingly, the geometries obtained through these initial optimizations correspond to local energy minima achieved in the initial adsorption stage. Thus, the thermodynamically most stable adsorption state is eventually reached only once some free-energy barrier is overcome. The required energy input can be provided by the kinetic energy in the MD simulations at finite average temperature (300 K in our case). Such MD runs lasted for 1 or 2 ns, depending on the system, and in general showed a monotonic decrease of the potential energy to an equilibrium state that was always followed for more than 500 ps. Subsequent energy minimizations of a large number of instantaneous snapshots provided the most stable adsorption states which maximize the surface interaction through a large molecular flattening. In the case of the triple helix of the collagen-like fragment, however, this procedure did not produce the most stable state: rather, it only yielded two almost parallel filaments lying on the surface, superimposed by the third one with an overall H shape. When we manually shifted longitudinally the latter filament so that it lay on the surface orthogonal to the other ones, a further MD run lasting
13852 J. Phys. Chem. B, Vol. 108, No. 36, 2004
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Figure 2. The arrangement of the protein fragments adsorbed on graphite in the most stable final adsorption state obtained after long MD runs and geometry optimizations of many instantaneous snapshots. The figure shows one albumin fragment7 (above, at left), the fibronectin module8 (above, at right), and the collagen-like fragment (below). The residue hydrophobicity is also shown with a false-color scale reported in the figure (red for hydrophobic and blue for hydrophilic residues),13 while the backbone is displayed in red for visual clarity.
for 1 ns showed that it did rearrange itself so as to be parallel. In this way, the system could best optimize the interaction energy of the filaments both with the surface and among themselves. In fact, the most stable geometry finally obtained after optimization of many instantaneous snapshots yielded a much more favorable final arrangement compared to the previously found H geometry, which corresponds to a robust local energy minimum. The increased stabilization of the parallel arrangement over the H-shaped one amounts to about 110 kJ mol-1, both in terms of the average potential energy within the last part of the MD runs and of the energy minima. Thus, the simple MD simulations do produce a viable structure, but not the most stable one. The most stable final geometries eventually achieved by the protein fragments on the graphite surface are shown in Figure 2 and display an overall conformation very close to that adopted in the last part of the MD runs (only one albumin fragment is reported in the figure for simplicity, since the other one is similar, despite the different hydropathy pattern). As expected, all the fragments arrange themselves so as to maximize the number of residues in contact with the graphite surface, hence the interaction energy. This feature leads to the formation of a monolayer of amino acids for the albumin and the collagenlike fragments. Such arrangement is fully independent from the presence of charged side groups, as we checked for the albumin fragment where neutral side groups were initially considered.7 On the other hand, the full spreading of the larger fibronectin module is hindered by the greater number of intramolecular interactions, obviously related to its larger size, but mainly by the presence of a few disulfide bridges that act as chemical cross-
links. In fact, by replacing the disulfide bridges with thiol groups, a much larger spreading than shown in Figure 2 is achieved, essentially leading again to a monolayer of amino acids at very long simulation times. We shall come back later to this issue. As for the interaction energy, here we limit ourselves to point out that in the albumin and the collagen-like fragment of Figure 2 we find Eint ) 3.71 MJ mol-1 and Eint ) 1.47 MJ mol-1, respectively (the other albumin fragment not shown in the figure7 has Eint ) 3.44 MJ mol-1). These values correspond to an average interaction energy of about 56 kJ mol-1 per residue in the albumin fragments (this value turns out to be valid both with charged and with neutral side groups) and of 43 kJ mol-1 per residue in the collagen-like fragment, all residues being essentially in contact with the surface. For the fibronectin module, we find Eint ) 3.90 MJ mol-1, thanks to the larger number of residues, but again an interaction energy of 57 kJ mol-1 per residue in contact with the surface. Therefore, we suggest that 57 kJ mol-1 is the effective value for the interaction energy of a residue in contact with a graphite surface obtained after averaging over the 20 natural amino acids that form the albumin fragments and the fibronectin module. As for the smaller value of the collagen-like fragment, it is due to its primary structure, consisting of the repeated triplet Gly-ProPro. In fact, this triplet is close to neutral, from the hydropathy viewpoint, or slightly hydrophilic, and it lacks the hydrophobic residues that strongly enhance the interaction energy with the hydrophobic graphite surface. A noteworthy feature of the final geometries consists of the ordered arrangement in parallel strands, which forms the main issue of the present paper. This pattern is best seen in the
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Figure 3. The same as in Figure 2 for the fibronectin module after replacing the disulfide bridges with thiol groups. This arrangement was obtained after a 4-ns MD run and subsequent geometry optimization.
albumin and in the collagen-like fragments, but it is also present in the fibronectin module, despite the intramolecular disulfide bridges. Here, some parallel strands do form on the graphite surface, while others arrange themselves in a plane almost orthogonal to the graphite surface, in part reminiscent of the native β-sheet structure. Still, they keep a rough parallelism with the former ones. If the intramolecular cross-links are removed as said before, the larger spreading and flattening of the molecule on the surface eventually obtained at very long times enhances the strand parallelism despite the lack of hydrogen bonds among them. This feature is clearly evident in the geometry displayed in Figure 3, showing that all the parallel strands lie on the surface. Accordingly, this nanoscale self-organization appears to be a general feature of proteins adsorbed on crystalline graphite provided it is not hindered by intramolecular crosslinks. In Figures 2 and 3, we also report the hydropathy index of the residues on a false-color scale (blue for hydrophilic and red for hydrophobic residues, while the backbone is depicted in red for visual clarity),13 showing that this feature is of relatively minor importance because of the cooperativity of the adsorption process. In fact, the driving force for the parallel ordering of the strands is due to a combination of dipolar and dispersion interactions (full electrostatic interactions involving integer charges are effectively absent because of their delocalization on the nonneutral side groups) but not to new interstrand hydrogen bonds. Neither the difference between fibrous and globular proteins nor the specific secondary structure of the latter ones appears to be highly relevant. On the other hand, we stress that no backbone hydrogen bonds, typical of the secondary structure, are ever present, either in the optimized geometries or during the last part of the MD runs. This finding conclusively rules out the presence of new β-sheets driven by the surface, while the simultaneous optimization of the surface and of the intramolecular interactions leads to a nanoscale backbone organization. We also add that the fragment mobility on the surface is relatively large, but the parallelism shown in Figures 2 and 3 is basically kept for a few hundreds of picoseconds in
Figure 4. The scalar products between the virtual bond vectors connecting the CR carbon atoms plotted as a function of their topological separation from the first one. The filled symbols apply to the most stable adsorbed state and the empty symbols to the isolated molecule in the native state. The upper panel applies to the albumin subdomain shown in Figure 2,7 the central panel to the fibronectin module,8 and the lower panel to the collagen-like protein fragment.
the MD runs through a cooperative motion (essentially, a bidimensional diffusion). The ordered organization of the residues can be characterized through the scalar products li‚lj, where li is the generic virtual bond vector connecting subsequent CR carbons, li, normalized by its length: li ) li/|li|. In Figure 4, we report the scalar products l1‚lj calculated in the most stable states, by taking i ) 1, that is, the first virtual bond, as a function of the topological separation with the other ones, j-1. In the final adsorbed state (filled symbols), the scalar products are clearly much larger and closer to (1 than in the native fragment (empty symbols), indicating a much larger parallel or antiparallel arrangement. Such a feature is best seen in the albumin and in the collagen-like fragment
13854 J. Phys. Chem. B, Vol. 108, No. 36, 2004 (upper and lower panel in Figure 4) but is nonetheless quite evident also in the fibronectin module (central panel). Here, the larger molecular size requires also some connecting strands to be orthogonal to the parallel ones, whence the values of the scalar products close to zero. This behavior is also clearly shown by the geometry reported in Figure 3, and even slightly enhanced (these results are not shown in Figure 4 for clarity), although in itself the scalar products do not discriminate between parallel bonds lying on the surface or on a perpendicular plane. As a final point, let us note that upon inspection of Figure 2 one may suspect that the limited size of the graphite planes could largely affect, and perhaps direct, the tightly ordered arrangement of the albumin fragment. However, we checked on larger planes that the strand ordering is indeed a purely intramolecular feature, thus effectively ruling out simulation artifacts due to surface end effects. Concluding Remarks In this paper, we report our simulation results about the absorption of some protein fragments on a hydrophobic graphite surface. Considering fragments of globular proteins with R-helices (two albumin fragments) or β-sheets (a fibronectin module) and of a fibrous protein (a collagen-like triple-helix polypeptide), we found in all cases a very large surface adsorption, with a pronounced parallel ordering. Conversely, no such ordering is found when the same fragments do adsorb on a hydrophilic surface of poly(vinyl alcohol), or PVA,14 which shows that the ordered arrangement is entirely due to graphite. Moreover, since at room temperature PVA is well below its glass-transition temperature (Tg ≈ 360 K), we initially carried out the simulations by keeping fixed the polymer surface. The observed lack of ordering suggests that the parallel arrangement found on graphite is not related to the surface rigidity. Conversely, we propose that it should be attributed to a combination of the surface hydrophobicity together with its crystallinity and its smoothness (PVA was essentially amorphous, in our simulations). On the other hand, at present we cannot assess the relevance of each surface feature for the simulated ordered pattern. To clarify this issue, we are currently carrying out analogous simulations on crystalline hydrophilic surfaces of TiO2 (rutile, anatase, and brookite) and on less hydrophilic polymer surfaces such as poly(ethylene terephthalate), to understand the relative importance of the surface hydropathy and atomic ordering. We also stress that we performed most simulations in an effective dielectric medium, but additional MD simulations were also carried out in the explicit presence of water. In this way, we could check that the final geometries are not greatly modified upon introduction of the water molecules, the main changes involving only the orientation of the side groups so as to maximize their hydration. Thus, we conclude that the ordered arrangements shown in Figures 2 and 3 are basically unaffected by the simulation medium. Despite the extensive experimental work carried out about protein adsorption on biomaterials (see for instance some recent papers in ref 15 and the review in ref 16), the ordering effect predicted here for relatively small fragments on graphite was not directly observed in practice, apart possibly from the β-sheet structure found for bovine serum albumin on chromium and
Raffaini and Ganazzoli molybdenum surfaces in ref 17. One reason is clearly that most works were concerned with other issues, such as the amount of adsorbed proteins, the strength of adsorption, the thickness of the adsorbed layer, the influence of the solution medium, and so forth, as a function of the surface nature or treatment. Another important reason is that most often proteins do show intramolecular disulfide bridges that hinder the full molecular spreading and an extensive surface ordering, as suggested by our results for the fibronectin module in Figure 2. As a word of caution, our simulations cannot conclusively rule out a further surface refolding of the proteins6 with the possible formation of a new secondary structure consisting of β-sheets (the so-called “native surface state” in the language of ref 5), possibly somehow assisted by water, that might form at much longer times, well beyond the current computer possibilities. However, we believe the eventual formation of real β-sheets to be most unlikely on the graphite surface, because no activation energy is essentially required for the ordered strands, and the loose parallel arrangement is consistently kept for the largest part of the MD runs (hundreds of picoseconds) with no indication of long-lived hydrogen bonds. In conclusion, the present results obtained with a fully atomistic model indicate a strong ordering effect of the ordered graphite surface, with a pronounced parallel arrangement of intra- or intermolecular strands irrespective of the protein native structure, but with no new secondary structure. Acknowledgment. This work was financially supported by MIUR (Italian Ministry for Instruction, University, and Research). References and Notes (1) Kasemo, B. Surf. Sci. 2002, 500, 656. (2) Nakanishi, K.; Sakiyama, T.; Imamura, K. J. Biosci. Bioeng. 2001, 91, 233. (3) Oscarsson, S. J. Chromatogr., B 1997, 699, 117. (4) Zhdanov, V. P.; Kasemo, B. Proteins: Struct., Funct., Genet. 1998, 30, 168. Zhdanov, V. P.; Kasemo, B. Proteins: Struct., Funct., Genet. 1998, 30, 177. (5) Zhdanov, V. P.; Kasemo, B. Proteins: Struct., Funct., Genet. 2001, 42, 481-494. (6) Castells, V.; Yang, S.; Van Tassel, P. R. Phys. ReV. E 2002, 65, 031912. (7) Raffaini, G.; Ganazzoli, F. Langmuir 2003, 19, 3403. (8) Raffaini, G.; Ganazzoli, F. Langmuir 2004, 20, 3371. (9) InsightII 2000, Accelrys Inc.: San Diego, CA, 2000; URL http:// www.accelrys.com/. (10) Dauber-Osguthorpe, P.; Roberts, V. A.; Osguthorpe, D. J.; Wolff, J.; Genest, M.; Hagler, A. T. Proteins: Struct., Funct., Genet. 1988, 4, 31. (11) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235; URL http://www.rcsb.org/pdb/. (12) Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; DiNola, A.; Haak, J. R. J. Chem. Phys. 1984, 81, 3684. (13) Engelman, D. M.; Steitz, T. A.; Goldman, A. Annu. ReV. Biophys. Biophys. Chem. 1986, 15, 321. (14) Raffaini. G.; Ganazzoli, F. Paper in preparation. (15) Petrash, S.; Liebmann-Vinson, A.; Foster, M. D.; Lander, L. M.; Brittain, W. J. Biotechnol. Prog. 1997, 13, 635. Sheller, N. B.; Petrash, S.; Foster, M. D.; Tsukruk, V. V. Langmuir 1998, 14, 4535. Wertz, C. F.; Santore, M. M. Langmuir 1999, 15, 8884; 2001, 17, 3006. Wagner, M. S.; Horbett, T. A.; Castner, D. G. Langmuir 2003, 19, 1708. (16) Nakanishi, K.; Sakiyama, T.; Imamura, K. J. Biosci. Bioeng. 2001, 91, 233. (17) Pradier, C. M.; Ka´rma´n, F.; Telegdi, J.; Ka´lma´n, E.; Marcus, P. J. Phys. Chem. B 2003, 107, 6766.
