Forced Desorption of Bovine Serum Albumin and Lysozyme from

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Forced Desorption of BSA and Lysozyme from Graphite: Insights from Molecular Dynamics Simulation Christian Mücksch, and Herbert M. Urbassek J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b05234 • Publication Date (Web): 15 Jul 2016 Downloaded from http://pubs.acs.org on July 17, 2016

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The Journal of Physical Chemistry

Forced Desorption of BSA and Lysozyme from Graphite: Insights from Molecular Dynamics Simulation Christian Mücksch and Herbert M. Urbassek∗ Physics Department and Research Center OPTIMAS, University Kaiserslautern, Erwin-Schrödinger-Straße, D-67663 Kaiserslautern, Germany E-mail: [email protected],Phone:+49(0)6312053022

∗

To whom correspondence should be addressed

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Abstract We use molecular dynamics (MD) simulation to study the adsorption and desorption of two widely different proteins – BSA and lysozyme – on a graphite surface. The adsorption is modeled using accelerated MD to allow the proteins to find optimum conformations on the surface. Our results demonstrate that the “hard protein” lysozyme retains much of its secondary structure during adsorption, while BSA loses it almost completely. BSA has a considerably larger adsorption energy compared to lysozyme, which does not scale with chain length. Desorption simulations are carried out using classical steered MD. The BSA molecule becomes fully unzipped during pull-off, while several helices survive this process in lysozyme. The unzipping process shows up in the force-distance curve of BSA as a series of peaks while only a single – or few, depending on protein orientation – force peaks occur for lysozyme. The maximum desorption force is larger for BSA than for lysozyme, but only by a factor of about 2.3.

Introduction When a protein approaches and adheres to a surface several outcomes of this process called adsorption are possible. The adsorbed protein can be in various conformation states from native to unfolded, depending on the hydrophilicity of the surface, charge distribution, surface topography and protein structure. 1 This nonspecific process and the following protein-cell interactions determine the surfaces biocompatibility. Graphite which is widely used in its pyrolytic form 2 as an implant material due to its physical properties 3 displays a high biocompatibility. Therefore, we study the adsorption and forced desorption of two very different proteins, BSA and lysozyme, from a graphite surface. While the adsorption of proteins on inorganic surfaces has been studied extensively using simulation in the past 4–11 considerably less research has been done on the forced desorption induced by pulling the protein with an AFM tip off the surface. 7,8 The equivalent bioadhesion experiments using desorption can be performed with the use of scanning force spectroscopy (SFS). 12,13 2


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The problems that are posed when simulating the adsorption process are well known and have been described in several publications. 10,14,15 (i) Long timescale of simulation are needed in order to accommodate the protein in its optimum conformation on the surface; 11 (ii) large simulation box sizes are essential to comprise all the water. Problem (i) can be solved by using accelerated MD (aMD). 16 For problem (ii) sometimes so-called implicit water simulations are performed where the water molecules are not included in the simulation, but only the dielectric constant of water and mostly not even the viscous drag exerted by it is included in the equations of motion of the protein. 9 It is not clear whether this simplification is adequate. 17 These difficulties become even larger when performing pull-off simulations. (i) In experiment, pulling velocities are of the order of 10−6 m/s, such that an extension of BSA to its full estimated chain length of about 210 nm would require simulation times of around 0.2 s. In consequence, all simulations are usually performed with much larger velocities, around 1–10 m/s. We note that recently 18 a scheme was proposed which incorporates aMD into a pull-off simulation. We refrained from using this scheme here, since its optimum realization requires the use of several pulling velocities, which is – for the case of BSA – beyond our present computational capabilities. (ii) Since during desorption the protein is strongly lengthened, correspondingly long simulation boxes are needed. This prevented up to now the simulation of the desorption of large molecules such as BSA. We chose two widely different proteins, BSA and lysozyme, for our study. These differ in several respects: (i) size, which can be attributed either to the number of amino acids or to the radius of gyration describing the protein extension; (ii) unfolding tendency or hardness which is defined by the protein’s internal stability with BSA being described as a “soft protein” and lysozyme as a “hard protein”. 19 In the present paper we perform adsorption simulations of BSA and lysozyme for 40 ns using accelerated MD on a hydrophobic graphite surface. We consider this simulation time as adequate for describing an initial adsorption on an implant surface with respect to the

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computational limitations that allow the following desorption simulations at pulling velocities of 10 m/s.

Simulation Methods

Figure 1: Orientation 1 of BSA above the graphite surface in 0.1 M NaCl. Nonpolar residues are shown in blue, neutral residues in green, and polar residues in red. Water molecules are not shown for clarity while sodium ions are shown in yellow and the chloride ions in cyan. For performing molecular dynamics simulations NAMD 2.9 20 was used together with the CHARMM27 force field 21 and the TIP3P water model. 22 Throughout all simulations, the temperature was set to 310 K and the pressure to 1 atm. Prior to both adsorption and desorption simulations, energy minimization and equilibration were carried out for 200 ps to stabilize the thermodynamic properties. A 2 fs time step was enabled by employing the SHAKE algorithm. 23 Periodic boundary conditions in combination with the particle-meshEwald (PME) routine 24 were used to handle the long-range electrostatics. The structures of BSA and lysozyme were taken from the protein data base. In the case of BSA we now used structure 4F5S 25 at pH 6.5 with a net charge of −17 e that was neutralized at 0.1 M NaCl. This setup above the graphite surface in a NaCl solution is shown in Fig. 1. In our previous work 26 we had to use a homology model since structural data was not available back then. Structural data 1AKI 27 for hen egg-white lysozyme at pH of 4.48 4


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with a net charge of +8 e was also neutralized at 0.1 M NaCl. Each of the proteins was simulated in 2 different orientations where orientation 2 was obtained from orientation 1 by a 90◦ rotation around an axis parallel to the surface. The initial orientations for the two proteins are shown in Fig. 2. From the total residue count of the structures (583 amino acids for BSA and 129 amino acids for lysozyme) one can estimate the protein chain length of the BSA structure to 210 nm while the lysozyme structure reaches about 46 nm. All of the snapshots were rendered using VMD 28 and Tachyon. 29

Adsorption Since hydrocarbon contamination is believed to be responsible for making graphite more hydrophobic 30,31 but we do not know exactly what kind of hydrocarbons are present on the surface (compare 32 ), we model the surface by using an effective hydrophobicity that leads to a macroscopic contact angle of 80◦ . This angle was observed in previous contact-angle experiments after 3h of exposure to air, 32 which we consider a typical time scale in adsorption experiment preparations. The graphite surface is described by the Lennard–Jones parameters of Rmin /2 = 1.763388 ˚ A as provided by Werder et al. 33 and a scaled ǫ = −0.056485 kcal/mol to achieve the desired hydrophobicity. Both proteins start the adsorption phase with a minimum distance of 6 ˚ A to the graphite surface. In the case of BSA the simulation box had dimensions of 379 × 435 × 102 ˚ A with a total of about 5 · 105 water molecules. Since lysozyme is much smaller, it was placed inside a simulation box of 207 × 237 × 74 ˚ A with a total of about 1 · 105 water molecules. To account for long-term adsorption phenomena the implementation of dual accelerated MD 34–36 in NAMD 37 was used as we did previously. 16 The acceleration parameters were adapted for the different proteins since they contain different amounts of residues and water molecules but were not varied between the two orientations.

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Figure 2: Initial orientations of BSA and lysozyme above the graphite surface. Orientation 2 was obtained from orientation 1 by a 90◦ rotation around an axis parallel to the surface. Nonpolar residues shown in blue, neutral residues in green and polar residues in red. The N-terminus is shown in yellow and the C-terminus in orange. Note that the scale is identical in all subfigures.

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Desorption In order to fully desorb the proteins from their adsorbed state on graphite very long simulation boxes in pulling direction were needed with lateral dimensions reduced to the sizes of the adsorbed state. For BSA these account to 211 × 211 × 2254 ˚ A with a total of about 3.3 · 106 water molecules, and for lysozyme to 90 × 90 × 443 ˚ A with a total of about 1 · 105 water molecules. These box sizes were estimated from the respective chain lengths of the unfolded protein structures which were ≈ 210 nm for BSA and ≈ 46 nm for lysozyme. Steered molecular dynamics (SMD) simulations with a desorption velocity of 10 m/s and a spring constant of 0.03 N/m typical of scanning force spectroscopy experiments were employed. The fast pulling velocity is needed to desorb the proteins with a reasonable computational effort, although SMD simulations for BSA required up to 5 · 105 CPU hours for a single orientation to complete. The simulation times ranged between 6 ns for lysozyme and 25 ns for BSA until complete desorption. As an attachment for the virtual cantilever the top nitrogen atom was selected as the pull group.

Results and Discussion Adsorption The adsorption process of BSA and lysozyme on graphite is simulated using accelerated molecular dynamics for a total of 2 · 107 time steps. Although the two proteins show a different adsorption behavior both start by maximizing their surface contact which is seen in the attractive van-der-Waals interactions with graphite in the left part of Fig. 3. The radius of gyration with its component parallel to the surface shown in Fig. 4 gives us an idea how much the protein is able to spread out on the surface. The initial orientation plays an important role as it decides on the flexibility of the protein for spreading on the surface. Our simulations show that lysozyme as a “hard protein”, meaning a high internal

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stability, 19 has little tendency to unfold and spread on the graphite surface which is also seen in the adsorption snapshots Fig. 5. Our results are in contrast to the work of Raffaini et al. 9 that demonstrated complete unfolding of lysozyme on graphite. The reason hereto might lie in the different surface parameters used, our accelerated explicit water model vs. the implicit inviscid water model used by Raffaini et al. in combination with minimization routines and mainly our reduced amount of orientations due to the computational demands of the pull-off simulations which resulted in different starting configurations for the adsorption process. BSA on the other hand shows quite a strong reduction in secondary structure (see Table 1) and a small but noticeable amount of spreading especially for orientation 1 (see Fig. 4). Also the adsorption snapshots in Fig. 5 in comparison with Fig. 2 show the reduced secondary structure and spreading across the graphite surface. There has recently been another molecular dynamics investigation on the adsorption of BSA on graphene by Vilhena et al. 38 showing minimal structural changes. A comparison with our previous work in an implicit inviscid water environment regarding the secondary structure content 26 shows that it is very similar to that studied here (see Table 1) using accelerated molecular dynamics. Therefore, we argue that the 150 ns simulations in explicit solvent performed by Vilhena et al. 38 might suffer from insufficient sampling. Even for a small protein like lysozyme it was shown that a simulation time of 300 ns using classical molecular dynamics can be insufficient to completely describe the adsorption process. 11 Furthermore, differences in the surface parameters might also cause an altered adsorption outcome. When comparing these two proteins regarding their adsorption phase it becomes obvious that BSA shows a higher affinity towards graphite than lysozyme, especially by the much increased amount of unfolding shown in Table 1 and Table 2 and the relative change in the protein’s radius of gyration, Fig. 4. It is known that protein stability in the vicinity of surfaces depends generally on several influence factors, among which are direct surface-protein interactions, water mediated interactions (depending on surface hydrophobicity) and entropic effects. 39 In our case, especially due to the different lengths and hydrophobic inner parts

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of the two proteins considered here, competing factors like intrapeptide vs. protein-surface interactions affect the adsorption outcome but complicate a detailed analysis. Interestingly, regarding surface interactions, we find a direct correlation regarding the area on which the proteins spread and the van-der-Waals interactions which both differ by a factor of about 7 between BSA and lysozyme. 0

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Figure 3: Van-der-Waals energies of BSA (top) and lysozyme (bottom) with the graphite surface.

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Figure 4: Evolution of radius of gyration – component parallel to the graphite surface.

Table 1: Secondary structure of BSA before adsorption (‘initial’) and at the end of the adsorption (‘ads’) and desorption (‘des’) simulations at zero van-der-Waals interactions, 2 · 107 time steps, calculated with DSSP. 40 after 2 · 107 time steps

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

69.5 % 1.9 % 0% 0%

Orientation 1 ads 23.5 % 1.7 % 0% 0.5 %

Orientation 2 ads 23.3 % 3.8 % 0.5 % 1.0 %

Orientation 1 des 0% 0% 0% 12.4 %

Orientation 2 des 1.3 % 0.1 % 0% 0%

Table 2: Secondary structure of lysozyme before adsorption (‘initial’) and at the end of the adsorption (‘ads’) and desorption (‘des’) simulations at zero van-derWaals interactions, 2 · 107 time steps, calculated with DSSP. 40 after 2 · 107 time steps

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

28.4 % 6.5 % 3.1 % 10.2 %

Orientation 1 ads 29.3 % 1.1 % 3.6 % 7.1 %

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Orientation 2 ads 26.7 % 2.3 % 0% 8.4 %


Orientation 1 des 10.7 % 4.5 % 0% 1.6 %

Orientation 2 des 19.4 % 1.9 % 0% 1.6 %

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Figure 5: Topview snapshots of BSA and lysozyme after 2 · 107 time steps of accelerated adsorption. The color coding is explained in Fig. 2. The scale is identical in all subfigures.

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Desorption The adsorbed protein structures where then subjected to an external force by means of steered molecular dynamics for up to 25 ns in the case of BSA and up to 6 ns for lysozyme until complete desorption did occur. We define complete desorption by zero Lennard-Jones interactions between protein and surface. Desorption lengths and desorption forces are listed in Table 3. The force distance curves in Fig. 6 show a similar pattern for both proteins: the first stage consists of a steep rise in force until the hindering secondary structure is unfolded and the mainly frictional forces of the solvent due to the fast pulling velocity are overcome to allow for further desorption. The high desorption forces for both proteins show that adsorption to the graphite surface is indeed quite strong. Interestingly, in the first stage between 0.5 to 1 ns of desorption all orientations show an increased interaction with the surface (see Fig. 3) which is induced by the external force acting on the surface-distant site of the protein, i.e., at the pull-group that allows the protein to cross an energetic barrier that was previously hindering further adsorption. BSA and lysozyme exhibit quite a different desorption behavior. BSA has spread out much more during adsorption and also due to its bigger size has more surface contact and therefore a stronger binding to the surface. Furthermore, BSA being known as a “soft protein” with a low internal stability 19 shows an almost complete unfolding during desorption also seen in the right part of Table 1. The unzipping of the BSA molecule during desorption can also be seen in Fig. 7 which shows the protein structures at their maximum desorption force. At full desorption BSA is unfolded to nearly its entire polypeptide chain length also shown in Table 3. Lysozyme on the other hand keeps at least some of its structure during the desorption process. Here, desorption occurs faster due to the smaller overall size and the reduced surface interactions. The force-distance curves for lysozyme in Fig. 6 also show less features than for BSA which is mainly due to the much shorter pulling distance. Interestingly, lysozyme has about a 4.5-fold shorter chain length than BSA but needs only about a tenth of BSA’s 12


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desorption length. The reduced adsorption and fast desorption process while maintaining at least some of its secondary structure fit to the picture of lysozyme being a “hard protein” with high internal stability. 19 The relatively high unfolding forces of lysozyme can be attributed to the viscous forces due to the high pulling velocity. Table 3: Desorption force, i.e. maximum force in the force-distance curves (Fig. 6), and desorption length i.e., distance between surface and pulled protein atom at instant of vanishing van-der-Waals interaction between protein and graphite surface.

desorption force desorption length

BSA Orientation 1 Orientation 2 1.390 nN 1.415 nN 172.3 nm 203.9 nm

lysozyme Orientation 1 Orientation 2 0.590 nN 0.606 nN 21.5 nm 17.4 nm

Conclusions We study the adsorption and forced desorption of two widely different proteins, BSA and lysozyme, from a graphite surface. We took care that all parameters in the simulation – such as water environment, neutralizing salt content, surface conditions, pulling velocities – were identical for both molecules. The adsorption is performed using aMD in an explicit solvent since protein adsorption is a process that requires advanced sampling due to metastable states occurring while the protein is adapting to the surface. That allows the protein to optimize its conformation on the surface and to reach a state of high adsorption energy; this is a necessary prerequisite to obtain a good starting conformation for the desorption simulation. Previous studies already showed the advantage of using aMD over classical MD in adsorption studies. 16 Our results after adsorption are in agreement with previous work using an implicit solvation. 26 Advanced sampling is here of key importance when comparing with recent work by Vilhena et al. 38 where for BSA only minimal structural changes could be observed. Comparing the adsorption of BSA and lysozyme, we find:

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Figure 6: Force-distance curves for BSA (top) and lysozyme (bottom) during the desorption process. The point of complete desorption is marked by a circle which is defined by zero van-der-Waals interactions between protein and surface.

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Figure 7: Snapshots of BSA and lysozyme at maximum desorption force in their different starting orientations (‘Or’). Note that the scale has changed between the BSA and lysozyme subfigures.

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• The secondary structure of BSA (only helices) is largely destroyed, while it survives to some extent in lysozyme. This feature justifies the distinction of “hard” vs. “soft” proteins introduced by Norde and Giacomelli. 19 • Analogously the radius of gyration of BSA increases considerably more for BSA than for lysozyme during adsorption. • As a consequence of the increased spreading of BSA on the surface, the adsorption energy of BSA is considerably (by a factor of around 7) larger than that of lysozyme. This demonstrates that the adsorption energy is not simply proportional to the chain length of the molecules but rather to the surface area over which the protein has spread. The desorption simulations were carried out using steered MD in its classical formulation 41,42 at a velocity of 10 m/s. We find that • BSA becomes almost fully unzipped during pull-off, while several helices survive this process in lysozyme due to its reduced surface interactions and higher internal stability. Again this justifies the notion of hardness of lysozyme as compared to BSA. • The (complete or incomplete) unzippping of the molecule also shows up in the forcedistance diagram of the desorption process. A consecutive series of force peaks accompanies the unzipping process in BSA, while only a single or few (depending on protein orientation) force peaks occur for lysozyme. • The maximum desorption force is larger for BSA than for lysoyzme, but only by a factor of about 2.3. This means that simple estimates of the desorption force based on chain length or adsorption area do not describe our results. Future studies will focus on the desorption process and investigate in particular its dependence on the pull-off velocity. By performing a sequence of simulations with varied velocity, aMD may be used to extrapolate the results to low velocities approaching those used in force spectroscopy experiments. 18 16


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Acknowledgement We acknowledge financial support by 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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Forced Desorption of Bovine Serum Albumin and Lysozyme from

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