Molecular Dynamics Simulation of the Adsorption of a Fibronectin

Langmuir 2004, 20, 3371-3378

3371

Molecular Dynamics Simulation of the Adsorption of a Fibronectin Module on a Graphite Surface† 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 September 22, 2003. In Final Form: February 6, 2004 We report atomistic simulations of the adsorption of a fibronectin type I module on a hydrophobic graphite surface. This module comprises only β-sheets, unlike the albumin fragments previously investigated by us which contained only R-helices (Raffaini, G.; Ganazzoli, F. Langmuir 2003, 19, 3403-3412). As done in the latter case, most simulations are carried out in an effective dielectric medium by energy minimizations and molecular dynamics (MD). Further optimizations and MD runs in the explicit presence of water are also performed to assess the stability of the geometries found and to describe the solvation of the adsorbed fibronectin module. The initial adsorption is accompanied by local rearrangements of the strands in contact with the surface, but the overall molecular structure is largely preserved. Much larger rearrangements take place at longer times as found through the MD runs, with the molecule spreading as much as possible so as to maximize the surface coverage, hence the interaction energy, despite a significant strain energy. Energetic aspects of adsorption together with the concomitant size change are discussed in comparison with our previous results for two albumin fragments.

Introduction The interactions of a foreign material in a living body, for instance, in a body implant, are typically mediated by proteins that may adsorb on the material surface. Beyond their theoretical relevance, such features are essential for assessing the material biocompatibility. Modeling such interactions for an ideal yet realistic case with atomistic simulations is the main goal of our current work. In recent years, computer simulations have added significant new insights to our understanding of protein adsorption on a foreign surface, complementing both theory and experiments. Such phenomena are not easily amenable to either analytical approaches or coarse-grained simulations because of the structural details at the atomistic level and of the rearrangements, or even denaturation, these molecules can undergo upon adsorption.1-7 Colloidal models yielded reasonable results from many viewpoints when electrostatic interactions with a charged surface are dominant,8,9 although all atomistic features, in particular the details of the electrostatic potential at the protein envelope, are ignored. More recent work explicitly considered the full protein structure to investigate the initial adsorption stage on a charged surface,10-12 † Dedicated to Professor Giuseppe Allegra on occasion of his 70th birthday. * To whom correspondence should be addressed. E-mail: [email protected].

(1) Nakanishi, K.; Sakiyama, T.; Imamura, K. J. Biosci. Bioeng. 2001, 91, 233-244. (2) Oscarsson, S. J. Chromatogr., B 1997, 699, 117-131. (3) Proteins at interfaces II: Fundamentals and Applications; Brash, J. L., Horbett, T. A., Eds.; ACS Symposium Series 602; American Chemical Society: Washington, D.C., 1995. (4) Buijs, J.; Norde, W.; Lichtenbelt, J. W. Th. Langmuir 1996, 12, 1605-1613. (5) Kim, D. T.; Blanch, H. W.; Radke, C. J. Langmuir 2002, 18, 58415850. (6) Petrash, S.; Liebmann-Vinson, A.; Foster, M. D.; Lander, L. M.; Brittain, W. J. Biotechnol. Prog. 1997, 13, 635-639. (7) Sheller, N. B.; Petrash, S.; Foster, M. D.; Tsukruk, V. V. Langmuir 1998, 14, 4535-4544. (8) Roth, C. M.; Lenhoff, A. M. Langmuir 1995, 11, 3500-3509. (9) Ståhlberg, J.; Jo¨nsson, B.; Horva´th, C. Anal. Chem. 1991, 63, 1867-1874; Ibid. 1992, 64, 3118-3124.

though still assuming a rigid molecular structure and ignoring any possible rearrangement to optimize the interaction. On the other hand, such rearrangements are quite common,1,2 particularly on hydrophobic neutral surfaces, as recently found experimentally.7,13,14 Coarsegrained lattice simulations were also used to investigate certain general aspects of protein folding15 or the kinetics of absorption, denaturation,16 and possibly refolding on a surface to a different secondary structure.17 However, it is clear that such methods oversimplify the protein behavior in that they completely neglect the local details and, in particular, all stereochemical features, which conversely can be of paramount importance. Therefore, they ignore the characteristics of the different amino acid side groups and usually the presence of the peptide moiety, which allows for the backbone hydrogen bonds (or H-bonds for short), hence for the secondary structure, even though some recent improvements have attempted to overcome in part the latter problem to study protein folding18 and surface adsorption.19 The local features are thus essential to properly understand protein adsorption on heterogeneous surfaces and can only be accounted for by atomistic simulations. Accordingly, we recently proposed a methodological approach to this problem in ref 20 (hereafter referred to as (10) Noinville, V.; Vidal-Madjar, C.; Se´bille, B. J. Phys. Chem. 1995, 99, 1516-1522. (11) Asthagirl, D.; Lenhoff, A. M. Langmuir 1997, 13, 6761-6768. (12) Ravichandran, S.; Madura, J. D.; Talbot, J. J. Phys. Chem. B 2001, 105, 3610-3613. (13) Wertz, C. F.; Santore, M. M. Langmuir 1999, 15, 8884-8894. (14) Wertz, C. F.; Santore, M. M. Langmuir 2001, 17, 3006-3016. (15) Dill, K. A.; Bromberg, S.; Yue, K.; Fiebig, K. M.; Yee, D. P.; Thomas, P. D.; Chan, H. S. Protein Sci. 1995, 4, 561-602. Pande, V. S.; Grosberg, A. Yu.; Tanaka, T. Rev. Mod. Phys. 2000, 72, 259-314. (16) Zhdanov, V. P.; Kasemo, B. Proteins: Struct., Funct., Genet. 1998, 30, 168-176 and 177-182. (17) (a) Zhdanov, V. P.; Kasemo, B. Proteins: Struct., Funct., Genet. 2001, 42, 481-494. (b) Castells, V.; Yang, S.; Van Tassel, P. R. Phys. Rev. E 2002, 65, 031912. (18) (a) Zhdanov, V. P.; Kasemo, B. Proteins: Struct., Funct., Genet. 1997, 29, 508-516. (b) Dimitrievski, K.; Kasemo, B.; Zhdanov, V. P. J. Chem. Phys. 2000, 113, 883-890. (19) Zhdanov, V. P.; Kasemo, B. Surf. Rev. Lett. 1998, 5, 615-634. (20) Raffaini, G.; Ganazzoli, F. Langmuir 2003, 19, 3403-3412.

10.1021/la0357716 CCC: $27.50 © 2004 American Chemical Society Published on Web 03/18/2004

3372

Langmuir, Vol. 20, No. 8, 2004

paper I). We considered two fragments of human serum albumin on the flat [0001] graphite surface and investigated the adsorption process through simple energy minimizations and molecular dynamics (MD) simulations taking into account all the molecular degrees of freedom. The hydrophobic graphite surface was chosen because of its relative simplicity and rigidity, so that it can be treated as a fully rigid body to all purposes. Albumin was selected because it is the most abundant blood protein, but the fibronectin modules are also important because they are involved in the late stages of the blood-clotting cascade. Therefore, in the present paper we apply the simulation procedure proposed in paper I to investigate the adsorption of a fibronectin type I module on a graphite surface. We follow again a two-step strategy:20 (i) first we carry out direct energy minimizations of the fibronectin module close to the graphite surface in different initial orientations, considering an effective dielectric medium that mimics water; (ii) then we perform MD simulations of selected geometries in the same medium and optimize many instantaneous snapshots in search of the most stable adsorbed state. As we have already suggested,20 the first step corresponds to the initial adsorption on a bare surface, but it could also provide the final stage at a large surface coverage in the absence of aggregation. Conversely, the second step yields the final adsorption stage with the largest interaction energy under thermodynamic control. The obvious computational advantages of adopting a dielectric medium should be noted. However, due to possible artifacts related with the strong hydration of polar and charged groups, we also carried out additional simulations by explicitly including a large number of water molecules to detect possible conformational changes induced by the solvent and to analyze the hydration pattern. In the following, after briefly summarizing the simulation method, we describe our results for the abovedescribed steps. In the final section, we summarize the results and compare them to those obtained for the albumin fragments, also providing an outlook to future work. Simulation Method All simulations were performed with the InsightII/Discover 2000 package, distributed by Accelrys Inc.21 (San Diego, CA), using the consistent valence force field22 CVFF with a Morse potential for the bonded atoms. This force field was chosen here, as in paper I, because it appears to be particularly efficient to deal with conformations and energies of peptides and proteins in general due to its parametrization. In this context, we note that CVFF describes nonbonded interactions through van der Waals and Coulombic terms only, with no extra terms for the hydrogen bonds. Moreover, it does not account for polarizability, just as the vast majority of force fields, which could be a possible limitation when dealing with a graphite surface. The coordinates of the non-hydrogen atoms used for the initial trial geometries were taken from the experimental results obtained by NMR experiments in solution and deposited with the Protein Data Bank (fibronectin type I module, 1FBR, ref 23), while the hydrogen atoms were added in the calculated positions. The molecule was then fully optimized up to an energy gradient lower than 4 × 10-3 kJ mol-1 Å-1. The graphite planes were prepared as described in paper I. The fibronectin module was then placed (21) Accelrys Inc. InsightII 2000; Accelrys Inc.: San Diego, CA, 2000. See also http://www.accelrys.com. (22) Dauber-Osguthorpe, P.; Roberts, V. A.; Osguthorpe, D. J.; Wolff, J.; Genest, M.; Hagler, A. T. Proteins: Struct., Funct., Genet. 1988, 4, 31-47. (23) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. The Protein Data Bank. Nucleic Acids Res. 2000, 28, 235-242. See also http://www.rcsb.org/ pdb/.

Raffaini and Ganazzoli

Figure 1. Experimental geometry of the fibronectin module. The secondary structure is indicated through the solid arrows indicating the β-sheets, while the ribbon indicates the backbone trajectory in the random strands and in the regular turns. close to the surface and optimized in an effective dielectric medium with a distance-dependent dielectric constant asymptotically yielding the water value ( ) 78). The energy minimizations in water were carried out by adding a few thousands of water molecules with periodic boundary conditions, adjusting the density to 1 g cm-3, and setting ) 1. The MD simulations were performed in the dielectric medium or in water with periodic boundary conditions at a constant temperature (T ) 300 K), controlled through the Berendsen thermostat. Integration of the dynamical equations was carried out with the Verlet algorithm with a time step of 1 fs, and the instantaneous coordinates were periodically saved for further analysis or geometry optimization. In all cases, the MD runs in the dielectric medium lasted for at least 1 ns. Within these runs, the total and potential energy showed an initial decrease, possibly with a few separate kinetic stages, and then fluctuated around a constant value, indicating achievement of the equilibrium state. Each frame collected during the MD runs was then minimized up to a gradient of less than 4 × 10-3 kJ mol-1 Å-1. Additional short MD runs with a few thousands of water molecules (with periodic boundary conditions and a density of 1 g cm-3) were used to analyze the distribution of the solvent around the molecular backbone through the pair distribution function (see below). These runs lasted for 10 ps, and the frames were saved every 80 fs. Data Analysis. The geometries periodically sampled in the MD runs can be usefully analyzed through the pair distribution function gij(r) (or PDF for short). This function gives the probability density of finding atoms j at a distance r from atoms i and is defined as

gij(r) )

d〈Nij(r)〉 FjdV(r)

where d〈Nij(r)〉 is the average number of times the j atoms are comprised in a spherical shell of thickness dr at a distance r from atoms i within an MD run and Fj is their bulk density. Thus, gij(r) yields the average local density of atoms j (normalized by their average bulk value) in the shell volume dV(r) at a distance between r and r + dr from atoms i, giving an immediate picture of the local density of j atoms due to specific interactions, for instance, in a coordination shell. In the following, the ij subscripts will be dropped for simplicity, since no ambiguity between the two sets of atoms may arise.

Results and Discussion The fibronectin type I module is a single-domain globular polypeptide consisting only of antiparallel β-sheets connected by amino acids present as regular turns or in a random conformation, as shown in Figure 1. More details on the secondary structure and of the amino acids comprised within the β-sheets are reported in Table 1, together with the hydropathy index calculated through the Kyte-Doolittle scale24 by summing the values of the constituent amino acids (more precisely, of their side (24) Kyte, J.; Doolittle, R. F. J. Mol. Biol. 1982, 157, 105-132.

Adsorption of a Fibronectin Module

Langmuir, Vol. 20, No. 8, 2004 3373

Table 1. Amino Acids and Secondary Structure of Fibronectin, with Hydropathy Indices β-sheeta 1st 2nd 3rd

first amino acid

last amino acid

no. of amino acidsb

hydropathy indexc

GLU17 MET28 ILE41 ASN50 THR56 THR63 LEU74

PRO22 CYS33 THR44 ASP51 SER57 LYS66 ILE77

6 6 4 2 2 4 4

-14.1 6.9 5.6 -7.0 -1.5 -6.3 7.3

a The β-sheets are sequentially numbered from left to right in Figure 1. b The total number of amino acids, 93, exceeds the sum of those in the β-sheets because of the presence of regular turns and random strands. c The hydropathy index was simply calculated as the sum of the values for the individual amino acids, from ref 24.

groups). It should be recalled that a positive hydropathy value indicates hydrophobicity and a negative one hydrophilicity. In Table 1, we see, for instance, that the β-sheets of the fibronectin module comprise strands of widely different hydropathy indices, although amino acids of unlike character are present in all cases anyways, irrespective of the overall index. We also add that the hydrophobic residues are all buried inside the molecule, while the outer envelope comprises only hydrophilic or at least non-hydrophobic residues, in keeping with the general pattern of globular proteins. We also generated one large graphite domain consisting of two carbon planes only, as already done in paper I,25,26 with a flat surface measuring 84 Å × 59 Å. Most simulations were carried out in a dielectric medium, based on our previous results for the albumin fragments.20 However, a few simulations were also performed in water to assess the overall stability of the adsorbed system and the extent of molecular hydration. It should be recalled that the optimizations in water cannot establish the relative stability of different adsorption geometries because the energy of the whole system is dominated by the overwhelmingly large contribution of the water molecules (of the order of a few thousands). Moreover, thermodynamically the energy minimizations correspond to freezing the system at 0 K with the water molecules in some glasslike configuration with a local energy minimum. In conclusion, the optimizations in water can show solventinduced conformational effects and the hydration pattern but not the system stability. 1. Initial Adsorption Stage in the Dielectric Medium. At first, we optimized the geometry of the isolated fibronectin module in the dielectric medium. The minimized energy, to be used later to calculate the interaction energy with the graphite surface, amounts to 3.13 MJ mol-1. As already found for the albumin fragments, there are relatively small differences between the optimized and the experimental geometries of the backbone but different orientations of the side groups due to the lack of hydration and a slightly larger number of intramolecular H-bonds. Afterward, we optimized the geometry of the fibronectin module close to the graphite surface, keeping fixed throughout the carbon planes. Since the module may be roughly inscribed in a rectangular prism (see Figure 2 at top), we considered six different starting orientations, corresponding to each face of the prism lying on the surface, as shown in the lower part of Figure 2. (25) Hentschke, R. Macromol. Theory Simul. 1997, 6, 287-316. (26) Mantero, S.; Piuri, D.; Montevecchi, F. M.; Vesentini, S.; Ganazzoli, F.; Raffaini, G. J. Biomed. Mater. Res. 2002, 59, 329-339.

Figure 2. Fibronectin module inscribed in a rectangular prism (top, see also text) and the six directions of close approach to the flat graphite surface according to the six faces of the prism (bottom). The two carbon planes are shown in a side view. Table 2. Results of the Initial Energy Minimizations and of MD Runs and Optimizations in the Dielectric Mediuma position 1 2 3 4 5 6 I II

Erel

Eint

Estrain

n5Å

0.83 0.13 0.81 0 1.28 1.70

initial minimizations 1.46 0.42 29 2.16 0.58 41 1.48 0.41 26 2.29 0.40 41 1.01 0.14 19 0.59 0.21 12

Hintrabroken

βAA

13 5 12 11 -1b 2

23 22 12 19 22 37

after MD runs and minimizations -1.12 3.41 0.64 53 14 -1.61 3.90 1.02 68 21

0 0

a We report the total energy relative to the lowest-energy state found in the initial minimizations, Erel; the interaction energy with the surface, Eint; the molecular strain energy, Estrain; the number of amino acids at a distance less than 5 Å from the plane, n5Å; the number of intramolecular H-bonds broken after adsorption, Hintrabroken; and the number of amino acids still found in β-sheets, βAA. All energies are in megajoules per mole (see text). b A negative value actually indicates formation of additional H-bonds.

Upon direct energy minimizations, all six orientations showed a significant initial surface adsorption, usually accompanied by some rearrangements of the interacting strands, though not as large as in the albumin fragments.20 We anticipate that all the corresponding energy minima turned out to be local ones, as it will be shown later through the MD runs and subsequent optimizations. The results of the initial optimizations are summarized in Table 2. The lowest energy minimum, corresponding to the most stable state found by this procedure (position 4 in Table 2 and Figure 2), amounts to 0.84 MJ mol-1, and the energies for the other minima are reported relative to this value. The corresponding geometry is reported in the upper part of Figure 3 (position 4 of Figure 2), while the lower part of the same figure shows the geometry of the highest energy minimum (position 6 of Figure 2). We should add, however, that the latter geometry of the present module is unrealistic for the whole fibronectin, since it would require it to extend through the surface.

3374


Figure 3. Geometries of the most stable (top) and less stable (bottom) states of the fibronectin module adsorbed on the graphite surface in the dielectric medium in the initial adsorption stage. The detailed chemical structure is also shown, omitting the hydrogen atoms for clarity.

Accordingly, it is of interest only for the isolated structure considered in this paper. Some intramolecular rearrangements are evident in the optimized geometry of the most stable initial adsorption state shown in the upper part of Figure 3. Such rearrangements basically consist of the loss of the β-sheets at the right-hand side and at the center of the molecule, although the corresponding strands do remain roughly parallel to one another and to the surface. On the other hand, very little intramolecular changes, if any, are found upon adsorption in the less stable state (lower part of Figure 3), so that the geometry of the fibronectin module and its secondary structure are essentially unchanged. In addition to the relative energy of the initial adsorbed state (Erel), Table 2 reports also the interaction energy, defined as Eint ) (Efree + Eplanes) - Etot, where Efree is the energy of the free, isolated molecule in the optimized geometry. According to this definition, Eint > 0 is the energy required to detach the adsorbed molecule from the surface and bring it back to the free state. Also, since the planes were kept fixed in the simulations, we have Eplanes ≡ 0, although of course all the carbon atoms do correctly interact with the protein atoms. We also define Estrain ) Efrozen - Efree, where Efrozen is the energy of the fibronectin module in the frozen geometry it adopts upon adsorption. Let us describe the rearrangements found through these initial energy minimizations. In all cases, the strands close to the surface locally optimize the interaction, but unlike what found for the albumin fragments, in most cases this process does not fully disrupt the secondary structure, as shown in Figure 3. The driving force consists of the favorable van der Waals interactions with the hydrophobic surface, mainly due to the hydrophobic or at least the less hydrophilic residues. Thus, Eint can be as large as 2.29 MJ mol-1 for the most stable orientation (Figure 3, top), to be compared with the value of 0.59 MJ mol-1 for the less stable adsorbed state (Figure 3, bottom). Considering that the fibronectin module is composed by 93 total residues, these values correspond to 25 and 6 kJ mol-1 per amino acid. We incidentally note that the former figure happens to be equal to what was found for the low-energy initial


Figure 4. Interaction energy Eint and strain energy Estrain of the fibronectin module (filled symbols) over the graphite surface after the initial energy minimizations in various orientations plotted as a function of n5Å, the number of amino acids being in contact with the surface at a distance less than 5 Å. The solid lines are the best-fit lines through the origin given by eqs 1 and 2. The correlation coefficients are R ) 0.9936 and 0.8319. For comparison, the analogous results obtained in ref 18 for two albumin fragments are shown with empty symbols and dotted lines.

adsorption stage of the albumin fragments.20 It is also interesting to point out that the Estrain roughly follows the same trend as Eint, amounting, for instance, to 0.40 and 0.21 MJ mol-1 for the above-mentioned geometries. Therefore, as already noted in paper I, a larger molecular deformation allows for stronger interactions with the surface that largely compensate for the greater strain energy mainly due to broken H-bonds of the β-sheets. As a consequence, a large Estrain is often accompanied by a small number of amino acids still structured in β-sheets (see Table 2). Stronger interactions are related with a larger number of residues in contact with the surface, irrespective of their hydropathy index, just as found in paper I, thanks to the apparently random distribution of hydrophobic and hydrophilic residues. Thus, we find a significant positive correlation between both Eint and Estrain and the number of amino acids in contact with the surface, n5Å, also reported in Table 2 (we conventionally take 5 Å as the upper limit for the contact distance with the surface). Such correlation is shown in Figure 4, where Eint and Estrain are plotted as a function of n5Å (filled symbols) in comparison with the values obtained in paper I for the albumin fragments (empty symbols). The best-fit lines through the origin for the fibronectin module are given by

Eint ) 54(1)n5Å kJ mol-1

(1)

Estrain ) 13(1)n5Å kJ mol-1

(2)

the figure in parentheses giving the estimated standard errors on the last significant digits. The numerical coefficients should be compared with those found for the albumin fragments, namely, 71(3) and 17(1) kJ mol-1, respectively. In other words, in the initial adsorption stage


Langmuir, Vol. 20, No. 8, 2004 3375

Figure 5. Two snapshots of the adsorbed geometries at equilibrium in the MD runs starting from the most stable initial adsoprtion stage (position 4, shown at the top of Figure 3).

the albumin fragments in contact with the surface do show a larger interaction energy than the fibronectin module because the latter has a more hydrophilic envelope, but they also undergo a larger strain because of an extensive local unwinding of the R-helices. Conversely, only minor readjustments are found in the fibronectin module, thanks to its different secondary structure, leading however to a smaller interaction energy for initial adsorption even in the most favorable orientation. As already stressed,20 the residues hydropathy is qualitatively less important because of the presence of unlike amino acids in all strands and of the cooperative nature of the adsorption process. From eqs 1 and 2 we see that Eint increases with the number of residues in contact with the surface much faster than Estrain. By extrapolation, the fibronectin module may optimize the interaction with larger deformations than reported in Figure 3 once an appropriate energy barrier is overcome. The energy input required to overcome this barrier can be achieved through MD simulations by the kinetic energy, as shown in the next section. 2. Final Adsorption by MD Runs and Energy Minimizations in the Dielectric Medium. Selected geometries obtained in the previous section were subjected to MD runs in search of the best adsorption geometry with the overall energy minimum. As done in paper I, we chose as starting points both the lowest- and the highestenergy geometries found by direct energy minimizations (positions 4 and 6 of Table 2, shown in Figure 3). When starting with the former geometry, the MD run yielded a smooth and relatively small energy decrease lasting for about 900 ps, before equilibrium was reached. This state, followed for an additional 600 ps, showed only minor reversible changes involving a continuous break up and formation of the β-sheets with little overall changes in the backbone trajectory, as shown in Figure 5 through two representative snapshots. Further energy minimizations of selected frames provided no other significant changes, and the final geometry is similar to the lower snapshot of Figure 5, lacking any well-formed secondary structure. Some details of this final geometry are reported as entry I in Table 2. A significantly different kinetic pattern is followed starting with the least stable geometry (bottom of Figure 3). In this case, the fibronectin module showed a fast tilting of its longest axis toward the surface, leading to a quick energy decrease within the initial 100 ps. Afterward, further readjustments with some molecular spreading,

Figure 6. Side and top view of the fibronectin module adsorbed on the graphite surface in the lowest-energy state after the MD runs and subsequent energy minimization. For clarity, the backbone is shown in red and the amino acid side groups in blue. Note the lack of any secondary structure.

lasting for about 400 ps, lead to the final equilibrium state, monitored for an additional 500 ps. Energy minimizations of many instantaneous snapshots showed little additional changes and yielded the most stable state (entry II in Table 2). A view of this final state is reported in Figure 6, showing that no secondary structure (β-sheets) is retained. A detailed kinetic study of the surface adsorption and spreading of the fibronectin module and of the albumin fragments studied in paper I shall be reported in a separate paper.27 The whole process leads to a strong interaction between the fibronectin module and the surface in the best adsorption state, with most residues in contact with the surface (see Figure 6 and Table 2). Moreover, no secondary structure is present anymore and most intramolecular H-bonds are broken (see again Table 2). This result is in qualitative agreement with what we found for the albumin fragments,20 which however can form a monolayer of amino acids evenly coating the surface, with full denaturation and complete disruption of the whole pattern of intramolecular H-bonds. In this context we note that one might expect the fibronectin module to form a similar monolayer, with an even larger spreading than predicted here. However, in addition to the intramolecular interactions still present (the residual H-bonds and the dipolar interactions), the network of disulfide bridges hinders a full molecular spreading. In fact, we carried out preliminary MD simulations on a very large graphite surface (measuring 110 Å × 89 Å) of the fibronectin module after cutting all the disulfide bridges and introducing -SH groups. We found an extensive flattening of the molecule on the surface, suggesting that eventually a monolayer of amino acids may indeed develop also in this case. The very large stability of the final equilibrium geometry achieved for the fibronectin module can be gauged by its relative energy (see entry II of Table 2). In this state, the interaction energy amounts to 3.90 MJ mol-1, a value much (27) Raffaini, G.; Ganazzoli, F. Manuscript in preparation.

3376


larger than what was found in the initial adsorption stage. This energy corresponds to 57 kJ mol-1 per amino acid in contact with the surface, solely due to the dispersion (or van der Waals) forces. This value is very similar to what was obtained for the initial adsorption (see eq 1 and Figure 4) and is equal to the average value of 56 kJ mol-1 found for the albumin fragments.20 This similarity is hardly surprising since both proteins are made up of the same 20 natural amino acids, even though in somewhat different ratios. Incidentally, we also note that the strain energy of the adsorbed fibronectin module in the most stable state (see again entry II of Table 2) amounts to 15 kJ mol-1 per residue in contact with the surface, surprisingly similar to the value of 17 kJ mol-1 found for the albumin fragments. We believe this similarity to be accidental, in view of the different secondary structure of the two proteins. The rearrangements observed in the MD run somewhat increase the size and anisotropy of the fibronectin module. In fact, the radius of gyration Rg increases from a value of 16.1 Å for the isolated molecule to 18.9 Å for the final state shown in Figure 6. However, the change in molecular anisotropy is more significant. The molecular shape is best described through the principal axes λi (i ) 1, 2, 3) obtained upon diagonalization of the radius of gyration tensor. For the isolated molecule, the principal axes, arranged in decreasing order, measure 14.3, 5.8, and 4.5 Å, consistent with the molecule being roughly inscribed in an elongated rectangular prism (upper part of Figure 2). Conversely, in the final adsorption stage (Figure 6) these axes become equal to 16.2, 9.4, and 2.7 Å. Thus, while the smaller axis, now perpendicular to the surface, is strongly shortened, the two other axes display a significant increase due to the surface spreading. This spreading can also be described through the change in contact area (the molecular footprint) between the initial and the final adsorption stage. The contact area is simply obtained through the difference between the surface area accessible to the solvent before and after adsorption. In turn, the accessible surface, or Connolly surface, is evaluated through a spherical probe of radius 1.4 Å, mimicking a water molecule rolling on the exposed van der Waals surface. In the initial adsorption stage, the contact area amounts to 477 Å2 for the lowest-energy minimum (position 4 in Table 2 and upper geometry in Figure 3), while for the less stable energy minimum it amounts to 142 Å2 (position 6 in Table 2 and lower geometry in Figure 3). After the MD runs and geometry optimization, the contact area increases to 758 Å2 in the former case (entry I of Table 2, with an arrangement similar to that shown in Figure 5 at bottom) and to 752 Å2 in the latter one, corresponding to the final best adsorption geometry (see Figure 6 and entry II of Table 2). Interestingly, these final contact areas are essentially equal, but the interaction energy is quite different because of the different number of amino acids in contact with the surface. In fact, the latter number is not strictly related with the molecular footprint because of possible voids trapped between the molecule and the surface. 3. Simulations in the Explicit Presence of Water. At first, the geometries previously obtained in the dielectric medium for the initial adsorption were further optimized in the presence of a large number of water molecules, showing only minor local changes. Therefore, the number of amino acids in contact with the surface, n5Å, is essentially identical to what was found in the dielectric medium. The small intramolecular rearrangements mainly involve some reorientation of the side groups, leading to the formation of the intermolecular H-bonds, but also some slight


Figure 7. Pair distribution function g(r) of the oxygen atoms of the water molecules around the backbone of the isolated molecule in water and in the final adsorption stage.

readjustment of the backbone, which however basically keeps its original trajectory. Therefore, the molecular size is unaffected by hydration, with both the radius of gyration and the principal axes increasing by less than 0.1 Å, whereas the secondary structure is slightly affected, with shorter β-sheets. In keeping with the unchanged molecular size, the accessible surface area A of the molecule exposed to the solvent (the Connolly surface) is hardly modified upon solvation. We recall that this surface area can be taken as proportional to the dispersive interaction energy with the solvent, but it is not related to the solvation energy because it does not account for the electrostatic potential, in particular for the H-bonds. Thus, there is no significant correlation between A and the number of intermolecular H-bonds, although of course a larger surface area still allows for a better hydration. In the present case, for the most stable geometry in the initial adsorption stage A is equal to 1.09 × 103 Å2 in the dielectric medium and to 1.11 × 103 Å2 in water. These values are to be compared to the much larger value of 1.73 × 103 Å2 for the isolated molecule, which can be approached by the solvent from all sides, unlike the adsorbed one. Interestingly, in the final adsorption stage achieved after the MD runs and geometry optimization (see the previous section), both the molecular size and the accessible surface area do not show significant variations in water compared to what was obtained in the dielectric medium, as found for the initial adsorption. Thus, in the most stable state shown in Figure 6 (entry II of Table 2), A is equal to 1.07 × 103 Å2 in the dielectric medium and 1.08 × 103 Å2 in water. Also, the molecular footprint undergoes a minor increase from a value of 752 Å2 in the dielectric medium to 773 Å2 in water. To achieve a better picture of the module hydration, we carried out short MD runs of the most significant geometries in water following the same strategy used in paper I (see also the Simulation Method section). Thus, we could study the average distribution of water molecules around the backbone through the pair distribution function g(r) (PDF). This function yields the probability density of finding water molecules (or of its oxygen atoms in the present case) at a distance r from the backbone, defined through the NCOCR atoms. In Figure 7 we show the PDF of water around the backbone of the fibronectin module for the isolated molecule and for the final adsorption stage depicted in Figure 6. For the isolated molecule, the peak at 2.8 Å corresponds to the first hydration shell while a second maximum due to the second shell is found at about 4 Å. The analogous maxima found in the final adsorption stage are shifted to shorter distances, namely, 2.5 and 3.6 Å, but the peak heights are much smaller. Accordingly, the final adsorption leads to fewer water molecules more tightly bound to the backbone compared to what was found


for the isolated module. This pattern is quite different from what we obtained for the albumin fragments20 and can be rationalized by two considerations. On one hand, it should be recalled that one side of the adsorbed molecule is not accessible to water, being in contact with the surface. On the other, the fibronectin module has a hydrophilic envelope, which favors the hydration of the isolated molecule, while the molecular rearrangements the module undergoes upon adsorption also expose the hydrophobic residues to water. Both features indicate a poorer overall hydration, concurring to decrease the value of the pair distribution function at a fixed r. Conversely, the isolated albumin fragments chosen in paper I had residues of either hydropathy index at the molecular envelope and therefore displayed a slightly poorer hydration of the fragments that was maximized upon full spreading on the surface. As a conclusion of this section, we point out that most results rely on the use of an effective dielectric medium to describe the adsorption geometries and the interaction energies, while the contribution of the solvation energy, specifically the hydration of polar and charged groups, is neglected both for the isolated and adsorbed module. As a word of caution, we note that neglecting these specific hydration effects may seriously affect both the kinetics of surface adsorption and its thermodynamics, i.e., the relative stability of the adsorbed geometries. The present simulation results must therefore be understood within the context of these conditions. Concluding Remarks In the present paper we investigate the adsorption of a fibronectin type I module on a graphite surface by atomistic simulations. The secondary structure of this module comprises only antiparallel β-sheets in addition to random strands and is therefore fundamentally different from the albumin fragments containing only R-helices previously studied by us.20 We first carried out direct energy minimizations of the fibronectin module in a dielectric medium close to the graphite surface in different orientations, thus obtaining the initial adsorption state. In this case, the strands close to the surface adjust themselves so as to optimize the interaction with the surface. However, unlike what was found for the albumin fragments, there is only a minor loss of secondary structure thanks to the β-sheets geometry. Subsequent optimizations in water of the resulting geometries do not bring about significant overall changes, apart from some readjustment of the side groups. As already found for the albumin fragments, the initial optimizations in the dielectric medium show many widely different energy minima. Therefore, we conclude that the configurational phase space of proteins adsorbed on a heterogeneous surface displays, in general, a rugged energy landscape, reminiscent of glassy states. However, the energy barriers separating the local minima are often not prohibitively high and can be easily surmounted through a suitable kinetic energy input. Accordingly, the most stable state for the fibronectin module on the graphite surface is readily found through molecular dynamics runs at room temperature. Depending on the initial adsorption geometry, we found two different outcomes. In the first case, we observed a relatively structured equilibrium state showing a continuous breaking and reforming of β-sheets through minor displacements of the backbone trajectory. This state produced a robust energy minimum with an overall geometry reminiscent of the isolated molecule. In the second case, we observed major rearrangements and a complete denaturation with a stronger adsorption. In

Langmuir, Vol. 20, No. 8, 2004 3377

the absolute energy minimum, found after optimization of many selected snapshots, the fibronectin module optimizes the interaction with graphite by spreading as much as possible and thus maximizing its footprint. We found a similar behavior for the albumin fragments, consistent with the extensive denaturation experimentally found for the whole protein on a hydrophobic surface (a C16 self-assembled monolayer).7,13 As already pointed out by us,20 cooperative effects are important in bringing the hydrophilic residues as close to the surface as the hydrophobic ones. In general, the latter residues show stronger interactions with the hydrophobic surface, but even the hydrophilic residues display a favorable interaction while often still being exposed to water. In this context, we should also point out that the present MD simulations cannot rule out further surface rearrangements possibly taking place at much longer times that may enhance the intramolecular interactions without basically affecting the surface interaction. This slow process was indeed suggested by Monte Carlo simulations through a coarsegrained lattice model17a that also predicted a final surface refolding to a different secondary structure, although possible artifacts due to the lattice cannot be ruled out. On the basis of the present simulations and of those involving the albumin fragments, we propose some general features of protein adsorption on the hydrophobic graphite surface. Thus, in the initial stage protein adsorption is qualitatively independent from the orientation or the nature of the approaching domain, i.e., of its hydropathy. The initial adsorption is accompanied by local rearrangements at the surface, possibly involving some loss of secondary structure, in particular in the case of R-helices, but with little change of the more distant parts. After this stage, the protein reorients and unfolds on the surface, spreading as much as possible with large-scale rearrangements, maximizing the number of residues in contact with the surface. These results are in fair agreement with recent experimental findings for albumin,6,13 while corresponding data for the fibronectin module studied by us are lacking to the best of our knowledge. However, it should be recalled that, in general, both hard and soft proteins do show an enhanced adsorption on hydrophobic surfaces with significant changes of conformations and denaturation.1,2 Some implications of this picture for albumin adsorption either on a clean or an already covered hydrophobic surface were discussed by us20 and can be carried over to the case of the fibronectin module. Thus, we expect, in general, that in the initial stage proteins may show reversible adsorption to some degree, unlike what happens in the final stage. In fact, adsorption irreversibility is consistent with experimental results on albumin6,7,13,14 and proteins, in general, on hydrophobic surfaces.1,2 The large molecular spreading on the surface also has obvious implications for adsorbed proteins under flow, similar to what we discussed in paper I. In particular, a small cross section due to large spreading together with a large interaction energy suggest a tight adsorption and a large resistance to shear stresses. Only for the initial adsorption stage on a partially covered surface these conclusions do not hold, and in such conditions adsorbed proteins might be easily removed under a large shear rate. As a final comment, we remark that in our description of the adsorption process of the fibronectin module (or of the albumin fragments) we implicitly ignored the possible adsorption and spreading of a second molecular layer on top of the first one consisting of the same or of different proteins or the possible replacement of one protein by a

3378


different one if the process is eventually under thermodynamic control. We hope to address such questions by appropriate simulations. Another relevant question is about the surface specificity, concerning its structure and rigidity and/or its hydrophobicity or hydrophilicity. Specific and lengthy experimental and simulation studies may help to answer such fundamental issues.


Acknowledgment. This work was financially supported by MIUR (Italian Ministry for Instruction, University and Research). Helpful discussions with Professor Giovanni Marletta and Dr. Laura Gambino are gratefully acknowledged. LA0357716

Molecular Dynamics Simulation of the Adsorption of a Fibronectin

Recommend Documents