Atomic-Level Study of Adsorption, Conformational Change, and

Jun 21, 2011 - The β-sheet conformation is not observed in all MD runs within the 15–200 ns times scale, which indicates that the α-helix to β-sh...
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Atomic-Level Study of Adsorption, Conformational Change, and Dimerization of an r-Helical Peptide at Graphene Surface Luchun Ou, Yin Luo, and Guanghong Wei* State Key Laboratory of Surface Physics, Key Laboratory for Computational Physical Sciences (Ministry of Education), and Department of Physics, Fudan University, 220 Handan Road, Shanghai, 200433, China

bS Supporting Information ABSTRACT: Recent circular dichroism spectroscopy and scanning tunneling microscopy study reported that a de novo designed R-helical peptide (with amino acid sequence DELERRIRELEARIK) would transform to β-sheet structure as well as random coil structure upon the addition of graphite particles to the peptide solution and aggregate into ordered βsheet-rich assemblies at the graphite surface. However, the atomic-level information about the dynamics of early stage conformational transition at water graphite interface and the driving force underlying the structural transition is largely unknown. In this study, we have investigated the conformational dynamics of two chains of the R-helical peptide in the absence and presence of a graphene sheet by performing all-atom molecular dynamic simulations in explicit solvent at 310 and 330 K. Our simulations show that consistent with the signal measured experimentally under physiological buffer conditions, two chains are mostly dimeric and keep R-helical structure in solution, whereas they unfold and assemble into an amorphous dimer at graphene surface. The β-sheet conformation is not observed in all MD runs within the 15 200 ns times scale, which indicates that the R-helix to β-sheet transition for this short peptide at graphite surface is a slow process, similar to the slow transition dynamics of globular protein reported experimentally. By analyzing all MD trajectories, we found that (1) the formation of R-helical dimer in solution is mostly driven by interpeptide hydrophobic interactions; (2) the adsorption and the R-helix unfolding of the peptide at graphene surface is initiated from the C-terminal region due to strong interactions between residues Arg13-Ile14-Lys15 and graphene surface; (3) the extent of helix unfolding strongly depends on the interaction strength between the peptide and graphene surface; and (4) the dimerization of two unfolded peptide chains at graphene surface results from the interplay between peptide graphene and peptide peptide interactions. This study would provide significant insight into the detailed mechanism of graphite-induced conformational transition and dimerization prior to the formation of β-sheet assemblies of this short synthetic R-helical peptide.

’ INTRODUCTION Protein surface interaction has drawn significant attention because of its important role in many fields,1 such as biosensors,2,3 biomedicine,4 bionanotechnology,5 and drug delivery.6 During the interaction process, surface may perturb or stabilize the native structure of proteins as well as the protein self-assembly pathway.7,8 A detailed mechanistic understanding of the protein surface interaction would be helpful to nanoscale materials design and bionanoassembly. Fundamentally, studies of protein surface interaction may help us to understand further the mechanism of protein folding, misfolding, and aggregation, which is related to recognized clinical disorders including Alzheimer’s disease, prion disease, and type II diabetes.9 These diseases are considered to be associated with protein conformational conversion from R-helix/random-coil to β-sheet.10,11 It is of fundamental importance to understand the conformational transitions triggering the amyloidogenesis. Accumulating data suggests that the effect of a surface on protein conformation and self-assembly depends on the chemical features of the peptide monomer, the way it interacts with the r 2011 American Chemical Society

surface, and the physicochemical properties of the surface (including hydrophobicity and charge).7,12 For example, experimental studies reported that on hydrophobic graphite surfaces, the Alzheimer’s amyloid β-peptide (Aβ) forms uniform, elongated β-sheet,13 whereas less-ordered particulate aggregates appear both at hydrophilic mica13 and positively charged surfaces.14,15 Recent Fourier transform infrared (FTIR) spectroscopy study showed that hen egg white lysozyme, when adsorbed at surfaces, exhibits R-helix to β-sheet transition over a long time (g1 min).16,17 Similarly, the adsorption of Aβ peptide on the surface of hydrogenerated nanoparticle induces a conformational transition from random-coil to β-sheet structure.18 Although these experimental studies have greatly enhanced our understanding of the impact of the surface on the protein conformation, the detailed adsorption and structural dynamics at surfaces are not well understood. Complement to Received: February 14, 2011 Revised: May 12, 2011 Published: June 21, 2011 9813

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Table 1. Setup Details of All MD Simulations and the Cr-rmsd of the Peptide at the End of Each MD Runa

a

The two peptide chains are labeled as P1 and P2, with the C-terminus being denoted by letter C. The carbon atoms of graphene sheet are in yellow. To mimic the experimental neutral pH condition, the side chains of Asp, Glu, Arg, and Lys are charged (Asp , Glu , Arg+,Lys+). All MD runs use different initial velocities. rmsd refers to the CR-rmsd of P1/P2 at the final simulation time of each MD run with respect to its conformation in the initial state of Pep system.

experimental studies, all-atom molecular dynamics (MD) simulations can provide detailed information on the interactions of proteins with surfaces and peptide conformational change. Therefore, computational studies on the protein adsorption at different surfaces at the atomic level have been emerging in recent years. For example, the adsorption dynamics of human lysozyme on hydrophobic graphite surface was investigated by 10 ns MD simulations in implicit solvent.19 The conformation changes of R-helices in the A subdomain of human serum albumin protein,20 40-residue polyalanine peptide,21 30-residue insulin chain B peptide,22 and short peptide HWSAWWIRSNQS23 on carbon nanotubes were studied by all-atom MD simulations in explicit solvent within a 10 ns time scale. The effect of hydrophobic and hydrophilic properties of surfaces on the adsorption of structured Aβ1-42 monomer and Aβ17-42 oligomer has been reported by Zheng et al.24,25 More recently, Chen’s group has studied the roles of different energy compositions of peptide surface and peptide peptide interactions on the adsorption of two randomly structured chains of an ionic complementary peptide EAK16-II (with sequence AEAEAKAKAEAEAKAK) at hydrophobic graphite surface by MD simulations in explicit solvent.26,27 It was found that the hydrophobic interaction is the main force to govern the peptide adsorption, and the interpeptide electrostatic interaction affects the

adsorption rate. Those MD studies, mostly within a 10 ns time scale (except refs 24 and 25), focus on the surfaced-induced R-helix unfolding of a single peptide/protein,19 23 the stability of monomer or oligomer with R/β content,19,24,25or the adsorption dynamics of two peptide chains with random coil character.26,27 A more thorough study that includes all three aspects has not been reported. The purpose of this work is to study at the atomic level the adsorption, conformational dynamics and the dimerization of two chains of a designed R-helical peptide at graphene surface using all-atom MD simulations up to a 200 ns time scale. The selection of this peptide is motivated by a recent experimental study using circular dichroism (CD) spectroscopy and scanning tunneling microscopy (STM), which reported that this R-helical peptide would transform to β-sheet as well as random coil structure upon the addition of graphite particles to the peptide solution and form ordered β-sheet assemblies at the liquid solid interface of graphite.28 Toward this end, we have carried out multiple MD runs with different simulation time (two 15 ns, four 60 ns, and one 200 ns) in explicit solvent at 310 K in the absence (A series of MD runs) and presence (B series of MD runs) of graphene surface starting from a state consisting of two parallel-placed R-helical chains (except for run B5). In addition, we have also performed two independent 9814

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Figure 1. Analysis of MD runs A1 and A2 for Pep system. (a) Time evolution of CR-rmsd of the two peptide chains (P1 and P2) with respect to the initial R-helical structure in Table 1. (b) Snapshot at the final time point of MD run. Time evolution of: (c) number of backbone H-bonds of peptide P1/P2, (d) fraction of initial R-helix of peptide P1/P2, (e) total number of interpeptide SC SC contacts, and (f) number of interpeptide HP SC SC contacts.

200 ns MD runs at 330 K in the presence of graphene sheet. The physical driving forces underlying the adsorption, conformational transition, and dimerization are also investigated.

’ MATERIALS AND METHODS The peptide has a length of two heptad repeats and consists of 15 residues, with amino acid sequence DELERRIRELEARIK, blocked by acetic acid (CH3CO) at the N-terminus and NH2 group at the C-terminus, as done experimentally.29 The initial state of the peptide system consists of two parallel-placed chains (Table 1), labeled as P1 and P2. The structure of the peptide is an R-helix, extracted from the crystal structure of four peptide trimers (PDB code: 1HQJ).29 To mimic the experimental neutral pH condition, the side chains of Asp, Glu, Arg, and Lys are charged (Asp , Glu , Arg+, Lys+). The graphene sheet is 5.1  5.1 nm2 in size, which provides a sufficient surface for the peptides to adsorb. The MD simulations are carried out in the isothermal isobaric (NPT) ensemble using the GROMACS software package30 and OPLS force field.31 Water molecules are realized by TIP3P model.32 The solute and solvent are separately coupled to

external temperature and pressure baths using Berendsen method.33 The temperature and the pressure are maintained at 310 K (or 330 K) and 1 bar using a coupling constant of 0.1 and 1.0 ps, respectively.33 Bond lengths within the peptides and water molecules are, respectively, constrained by the LINCS34 and the SETTLE algorithms.35 This allows an integration time of 2 fs. Particle mesh Ewald (PME) method is used to calculate the electrostatic interaction.36,37 van der Waals interactions are calculated with a cutoff of 1.4 nm. All MD simulations are performed using periodic boundary conditions in a rectangular box with a size of 5.1  5.1  5.0 nm3. The graphene is put at the edge of the 5.1  5.1 wall of the water box, and its position is fixed during the simulation. Carbon atoms of graphene sheet are uncharged in accordance with Hummer et al.38 The Lennard-Jones parameters are obtained using the Lorentz Berthelot rule39 to describe the protein graphene and water graphene interactions. The setup details of all the MD runs are given in Table 1. The two systems without and with graphene are labeled as Pep and Pep+Gra, respectively. The initial center-of-mass (COM) distance between P1 and P2 is 2.0 nm in both Pep and Pep+Gra systems. In the Pep +Gra system, the backbones of P1 and P2 are both parallel to the 9815

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Figure 2. Overview of the simulation results of MD runs B1∼B4 for the Pep+Gra system. Time evolution of CR-rmsd of P1/P2 with respect to the initial R-helical structure in MD runs: (a) B1 and B2 and (b) B3 and B4. (c) Snapshots at t = 15 ns in runs B1 and B2, where P1 and P2 are adsorbed on two different graphene surfaces. (d) Snapshots at t = 60 ns in B3 and B4, where P1 and P2 are adsorbed on the same graphene surface. Letter C represents the C-terminus of the peptide. The carbon atoms of graphene sheet are in yellow.

graphene sheet. The initial minimum (MIN) distance between P1/ P2 and the graphene surface (or the wall of water box) is 1.2 nm; that is, peptides and graphene are not contacted in the beginning of the simulations. The two A series 60 ns MD runs (A1 and A2) for Pep system start from the same initial state using different initial velocities. The first four B series of MD runs (B1∼B4) at 310 K start from the same Pep+Gra position coordinates using different random seed to generate initial velocity. The initial Pep+Gra state for run B5 is the configuration generated at t = 15 ns in run B4, in which the two chains are partially unfolded (see Table 1). The two MD runs (B6 and B7) at 330 K use the same initial states as those in B1∼B4 but different initial velocity distributions Trajectory analysis is performed using our in-house codes and the facilities implemented in the GROMACS software package.30 The adsorption dynamics of the peptide chain P1/P2 is analyzed by the time evolution of the MIN distance between the N-/Cterminal residues Asp1/Lys15 and the graphene surface (Asp1Gra, Lys15-Gra), the distance of the P1/P2 COM to graphene surface (P1-Gra COM, P2-Gra COM), and the MIN distance between P1/P2 and graphene (P1-Gra MIN, P2-Gra MIN). The conformational dynamics of the two chains is investigated by the time evolution of the CR-root-mean-square deviation (rmsd) with respect to the initial R-helical structure, the number of intrapeptide backbone hydrogen bonds (H-bonds), and the fraction of initial R-helix. The secondary structure content is identified using the DSSP program.40 The interpeptide hydrophobic (HP) interactions are analyzed by the number of sidechain—side-chain (SC SC) contacts of residues Leu3, Ile7, Leu10, and Ile14. The interpeptide interactions are evaluated by

the total number of SC SC contacts between the two chains. Here a contact is defined when aliphatic carbon atoms of two side-chains come within 0.54 nm or any other non-hydrogen atoms lies within 0.46 nm.41,42 A H-bond is considered to be formed if the donor acceptor distance is e0.35 nm and the donor hydrogen acceptor angle is g150°. All representations are prepared using VMD program.43

’ RESULTS AND DISCUSSION Two Peptide Chains Are Mostly Dimeric and Keep r-Helical Structure in Solution. Prior to studying the confor-

mational dynamics of two peptide chains at graphene surface, we characterize the structural dynamics of the peptides in solution by performing two independent 60 ns MD runs (A1 and A2) in the absence of graphene surface. The rmsd quantities of P1 and P2 at t = 60 ns are given in Table 1, and they are smaller than 0.2 nm, indicative of minor structural change with respect to the initial R-helical structure. Figure 1 presents the time evolution of several parameters of the two chains (P1 and P2) in runs A1 and A2. Figure 1a shows that the CR-rmsd’s of P1 and P2 in Run A1 are smaller than 0.15 nm in the first 30 ns; then, the rmsd of P2 stabilizes at 0.1 nm within the present 60 ns time scale, whereas the rmsd value of P1 increases to 0.2 nm at t = 30 ns, drops to 0.1 nm at t = 55 nm, and increases again to 0.2 nm at the end of the simulation. This change of the rmsd value corresponds to a local R-helix unfolding refolding of P1 during the last 30 ns of MD run. The CRrmsd’s of the two chains in run A2 both stabilize at 0.1 nm during 9816

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Figure 3. Detailed analysis of MD runs B1 and B2, where peptides P1 and P2 are adsorbed on different graphene surfaces. Time dependence of the MIN distance of the N-/C-terminal residues Asp1/Lys15 to graphene surface in MD runs: (a) B1 and (b) B2. Time evolution of: (c) fraction of initial R-helix of peptide P1/P2 and (d) the interaction energy between peptide P1/P2 and graphene.

the whole duration of 60 ns MD simulation. The small rmsd value in Figure 1a indicates that the two chains are stable in Rhelical structure in solution, as seen from the snapshots at t = 60 ns in Figure 1b. The structural stability of P1 and P2 is further illustrated by the time evolution of the number of backbone H-bonds (Figure 1c) and the fraction of initial R-helix (Figure 1d). The number of H-bonds for P1 and P2 is ∼9, with fluctuations between 7 and 10, and the two chains both keep over 70% of their initial helix content during the MD simulations. The stability of the R-helix observed in our MD simulations is in good agreement with the measured CD signal on this de novo designed polypeptide.28,29 The fluctuation of number of H-bonds corresponds to a break and reformation of intrahelical backbone H-bonds. A similar extent of fluctuation is also seen from the time evolution of fraction of initial helix, in which a partial unfolding refolding of R-helix takes place, indicating that the helical structure is dynamically stable in solution. To explore the interaction between P1 and P2, we plot the time evolution of the total number of SC SC contacts and the number of HP SC SC contacts in Figure 1e,f. It can be seen from Figure 1e that there are only a few interpeptide SC SC contacts at t = 0 ns, which is indicative of weak interactions between the two peptide chains in the initial state. The total number of SC SC contacts in run A2 increases rapidly and reaches to 220 within the first 5 ns, corresponding to a formation of an R-helix dimer. The following step is the rearrangement and the optimization of the nonhydrophobic side-chains at the dimer interface, which results in a significant decrease in the total number of SC SC contacts from 220 to 150, within the period of t = 5∼30 ns. Finally, we see a small fluctuation of contact number around 150 during the last 30 ns. In a different fashion,

the number of HP SC SC contacts reaches 60 at t = 5 ns and remains around that value in the left 55 ns MD run (see Figure 1f). These data indicate that the formation of a wellorganized HP core occurs prior to the well-packing of the nonHP side-chains at the dimer interface. Similar result is also obtained in run A1. These results suggest that the helical dimer formation is mainly driven by the interpeptide HP interactions, and the dimer may be stabilized by both hydrophobic and hydrophilic (including electrostatic) SC SC interactions. Previous experimental studies reported that hydrophobic interaction is a dominant force in stabilizing the parallel helical dimer.44,45 Peptide Graphene Interaction Induces r-Helix Unfolding and the Interpeptide Interaction Facilitates the Dimerization. Having now established that the force-field and simulation methodology used for the synthetic 15-residue peptide correctly yield an R-helical dimer being the stable state in solution within the present 60 ns time scale, we turn to the study of the conformational dynamics of two peptide chains at graphene surface using the same peptide force field and the MD approach. To this aim, we first performed four independent MD runs (B1∼B4) at 310 K starting from the initial state given in Table 1 using different initial velocity distibutions, in which the initial conformations of the two peptide chains are the same as those in the Pep system. In Table 1, we present the CR-rmsd of each chain at the end of each MD run. It can be seen from this Table that the rmsd of at least one peptide chain in each MD run is much larger than the rmsd in runs A1 A2, demonstrating that the presence of graphene surface perturbs the peptide structure and causes large conformational change. The detailed dynamics of this structural change can be seen from the time evolution of the rmsd of P1/P2 in Figure 2a,b. It is noted that the small rmsd 9817

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Figure 4. Detailed analysis of MD runs B3 and B4, where peptides P1 and P2 are adsorbed on the same graphene surface. Analysis of run B3: time evolution of: (a) the distance of the peptide COM to graphene surface and the MIN distance of P1/P2 to graphene surface; (b) interaction energy between P1/P2 and graphene; and (c) the MIN distance of the N-/C-terminal residues Asp1/Lys15 to graphene surface. The same analysis for run B4 is presented, respectively, in panels d f.

of P2 in runs B2 and B3 corresponds to weak interaction of P2 with graphene surface (see below for more detailed discussion). Figure 2c,d gives the snapshots at the end of the four MD runs, from which we see that two peptide chains partially or completely lose their helical structure. We also found that P1 and P2 adsorb either on two different graphene surfaces (Figure 2c) or on the same graphene surface (Figure 2d) because of the different initial velocity distributions used in the different runs. In the first case, the two chains are monomeric and do not have any atomic contacts (Figure S1 in the Supporting Information), whereas in the second case, they form an amorphous dimer via peptide peptide interactions. This observation indicates that the initial peptide peptide distance used in this study, which allows the two chains to be in either monomeric or dimeric state depending on the initial velocity distributions, does not highly influence the dimerization. In the following, we will investigate separately the peptide graphene interactions and the accompanied conformational changes in these two cases and focus on the second case as peptide dimerization occurs. The details of the interaction between peptide monomer and graphene surface (runs B1 and B2) are examined from the time dependence of the MIN distance between the two terminal residues (Asp1 and Lys15) and graphene surface (Figure 3a,b). It can be seen that the MIN distance of Lys15-Gra for P1/P2 reaches to ∼0.25 nm in the first 5 ns and remains almost constant in the left period of simulation, whereas the MIN distance of Asp1-Gra is usually >0.5 nm during the whole period of MD simulations (except for P1 in run B2, reaching to 0.25 nm at t = 11 ns). These data display that peptide adsorption at the graphene surface is mostly initiated from the C-terminal region of the peptide. During the adsorption process, the monomers in the two runs lose their initial R-helix content at different extents (see Figure 3c). By comparing Figure 3c,d, we found that the fraction of initial R-helix is strongly correlated with the peptide

graphene interaction energy, indicative of an adsorption-induced R-helix unfolding. Interestingly, the time dependence of the peptide graphene interaction energy for the two monomers exhibits a stepwise feature (Figure 3d), reflecting a stepwise adsorption dynamics of the peptide at graphene surface. By trajectory visualization, we found that each step in Figure 3d corresponds to a local rearrangement of the residues near the surface. The stepwise adsorption behavior was also proposed in a 2 ns MD study for the adsorption of A subdomain of human serum albumin on the surface of carbon nanotubes.20 However, we observe that the stepwise adsorption dynamics occurs on a much longer time scale, and the dwell time in one single step can last for 10 ns (see the green and blue curves in Figure 3d). To investigate the detailed peptide graphene interactions in the dimer system (runs B3 and B4), we plot in Figure 4 the time evolution of the MIN distance, the COM distance, and the interaction energy between P1/P2 and graphene surface as well as the time dependence of the MIN distance of Asp1-Gra and Lys15-Gra. As seen in Figure 4a,d, the MIN distances of P1-Gra and P2-Gra both decrease rapidly and drop to 0.25 nm in the first 5 ns. Differently, the distance between the COM of each chain and graphene surface reduces gradually in a stepwise fashion, reaching to 0.7 nm for three out of four chains within a 60 ns time scale. At each step, rearrangement of residues takes place to optimize the peptide surface interactions. It is notable that the P2-Gra COM distance in B3 is 1.2 nm at t = 60 ns, which implies that for the most part, chain P2 is not adsorbed on graphene surface, consistent with the weak P2 graphene interaction in Figure 4b. It is expected that a complete adsorption of P2 at graphene surface might be observed in a much longer MD simulation. The variation of graphene peptide interaction energy (Figure 4b,e) with time exhibits the same stepwise behavior as that of the graphene peptide COM distance (see Figure 4a,d). The dwell time of the peptide in a single step can last for 20 ns 9818

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Figure 5. Further analysis of MD runs B3 and B4. Time evolution of: (a) fraction of initial R-helix of peptide P1/P2, (b) number of backbone H-bonds of P1/P2, (c) total number of interpeptide SC SC contacts, and (d) number of interpeptide HP SC SC contacts.

before getting to the final step, much longer than the dwell time during monomer adsorption. Similar to the peptide adsorption behavior observed in Figure 3a,b for the monomer system, the time evolution of the MIN distance of Asp1-Gra and Lys15-Gra in Figure 4c,f shows that the C-terminal residue Lys15 has a preference to adsorb on the graphene surface. After characterizing the adsorption dynamics of the two chains P1 and P2, we investigate their conformational dynamics and the details of peptide peptide interactions. Figure 5a displays that P1 in run B3 loses almost 50% of its initial R-helix content, whereas P2 mostly stays in R-helical structure. By comparing the fraction of initial helix of peptides in Figure 5a with the P1/ P2 Gra interaction energy in Figure 4b,e, we see that P2 in run B3 keeps over 80% of its initial helix content, and its interaction energy with graphene surface is 100 kJ/mol, much weaker than the P1 Gra interaction in run B3 and P1 /P2 Gra interaction in run B4. The observed high percentage of helical structure of P2 in run B3 and the corresponding weak interaction of P2 with graphene sheet indicates that the intactness of R-helical structure of P2 is due to its weak interaction with graphene surface. This is consistent with recent CD spectroscopy study on the same peptide showing that the peptide adopts R-helical structure when it is unaffected by graphite particles.28 In run B4, P1 and P2 both have 90% of their R-helix content, whereas the N-terminal residues Glu2∼Arg5 still have 35 50% of R-helix content left. This observation demonstrates that the unfolding of the R-helix is initiated from the C-terminal region of the peptide. It can be seen from Figure 6b that the interaction strength of the C-terminal residues Arg13, Ile14, and Lys15 with graphene surface is ∼45 kJ/mol, much greater than the interaction strength of 9 20 kJ/mol between the N-terminal residues and graphene. By comparing Figure 6a,b, we find that the greater the residue graphene interaction strength, the more the loss of R-helical structure. This finding reveals that R-helix unfolding is mainly driven by the peptide graphene interactions. In addition, Figure 6b indicates that the adsorption preference of the C-terminus to graphene surface observed in Figures 3a,b and 4c,f is attributed to the strong interactions of the C-terminal residues with the uncharged graphene surface. Because the side-chains of residues Arg13 and Lys15 contain the alkylene chain segment ( (CH2)n ) and residue Leu14 has an aliphatic side chain, they

could be easily adsorbed at graphene surface because of van der Waals and hydrophobic interactions. At the same time, the strong residue graphene interactions would perturb the native intrapeptide interactions, thus inducing R-helix unfolding starting from the C-terminal region. Recent experimental study using CD spectroscopy and STM reported that this synthetic R-helical polypeptide would transform to β-sheet structure upon the addition of graphite particles to the peptide solution and form ordered β-sheet assemblies at liquid solid interface of graphite.28 However, β-sheet conformation is not observed in the four MD runs for the Pep+Gra system within the present 15/60 ns time scale. To see whether βsheet structure can form in a longer MD simulation, we have performed a 200 ns MD run (B5) at 310 K using a partially unfolded R-helix turn conformation generated at t = 15 ns in B4 as a starting state (see Table 1). Figure 7 gives the time evolution of the CR-rmsd and the secondary structure profile of P1/P2. The rmsd value of P1 fluctuates around 0.2 nm in the first 85 ns, then increases gradually with time and reaches to 0.25 nm at t = 115 ns (see Figure 7a). This rmsd increase is accompanied by a complete loss of R-helix remained in P1, as seen in Figure 7b. During the last 85 ns of the simulation, the rmsd of P1 fluctuates around 0.25 nm, and the peptide stays in a metastable turn bend conformation with partial 310-helix content. The rmsd of P2 fluctuates around 0.3 nm during the whole 200 ns MD simulation. The R-helix remained in its initial state is completely lost at t ≈ 35 ns; then, P2 adopts a turn bend conformation with 9820

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The Journal of Physical Chemistry B 310-helix content in the N-terminal residues Glu2∼Arg5. Although a complete unfolding of R-helix occurs for both P1 and P2, β-sheet conformation is not observed on the 200 ns time scale. To see whether R-helix to β-sheet conversion can be observed by raising the simulation temperature, we have performed two independent 200 ns MD simulations (B6 and B7) at 330 K using the same initial state as that of B1∼B4 MD runs. The results are shown in Figure S3 of the Supporting Information, which gives the time evolution of DSSP and CR-rmsd of each peptide chain and the snapshot of the dimer at t = 200 ns. A rapid R-helix f turn/coil transition is observed in both MD runs (Figure S3a,d of the Supporting Information); however, β-sheet structure is still not observed. The fast unfolding of R-helix observed here is consistent with previous experimental and computational data showing that raising the temperature would increase the helix f coil transition rate.47,48 It is noted that although β-sheet structure is not formed in all MD runs, isolated β-bridge is observed (Figure S2 of the Supporting Information, Figure 7b, and Figure S3a of the Supporting Information), albeit with a very low population. The formation of isolated bridge is the first step of β-sheet formation. These results, together with the result obtained from B5, indicate that β-sheet formation for this small peptide at graphene surface is a slow process. Fast loss of initial R-helix and slow dynamics of the R-helix-to-β-sheet transition for globular protein at surfaces has also been reported by experimental studies using FTIR spectroscopy.16,17 The experimentally probed R-helix-to-β-sheet transformation28 is likely to be observed on a microsecond time scale by constant-temperature MD simulations, which is still out of reach using current computing facilities.49

’ CONCLUSIONS We have systematically studied the adsorption, conformational dynamics, and the dimerization of two chains of a de novo designed R-helical peptide at graphene surface prior to the formation β-sheet assemblies by carrying out all-atom molecular dynamic simulations in explicit solvent within a 15 200 ns time scale. Our simulations show that the C-terminal residues have a preference to adsorb at graphene surface because of the strong interactions of residues Arg13, Ile14, and Lys15 with graphite surface, leading to a fast unfolding of R-helix starting from the C-terminal region. The peptide adsorption dynamics exhibits a stepwise feature, and the dwell time of the peptide in one single step during dimerization can last for 20 ns before getting to the final step, much longer than the dwell time during monomer adsorption. Our calculation shows that the extent of R-helix unfolding strongly depends on the interaction strength between the peptide and graphite surface. This result provides direct evidence of an adsorption-induced conformational change. By analyzing the peptide graphene and peptide peptide interactions, we found that peptide graphene interactions would perturb the native intrapeptide and interpeptide interactions, thus inducing R-helix unfolding. The formation of amorphous dimer by unfolded chains at graphene surface results from the interplay between peptide graphene and peptide peptide interactions. During the unfolding process, a conformation conversion from R-helix to 310-helix is observed in our MD runs. The 310-helix content has also been detected during the amyloid fibril formation process of a 15-residue R-helical peptide (AEQLLQEAEQLLQEL) in bulk solution by 2D-NMR, CD,

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and FTIR spectroscopy.46 This study, complement to the recent experimental study,28 provides at the atomic level a detailed dynamics of adsorption-induced secondary structure transformation and dimerization of an R-helical peptide at the graphene surface. The R-helix to 310-helix/turn/bend transition and the formation of amorphous dimer may play an important role in the aggregation of the peptides into β-sheet assemblies.

’ ASSOCIATED CONTENT

bS

Supporting Information. Time evolution of total number of interpeptide SC SC contacts in MD runs B1 and B2, time evolution of secondary structure profile of chain P1/P2 in run B4, and trajectory analysis of two independent 200 ns MD runs (B6 and B7) at 330 K. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Tel: (86) 21 55665231. Fax: (86) 21 65104949. E-mail: ghwei@ fudan.edu.cn.

’ ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (grant no. 11074047), the Program for New Century Excellent Talent in University (NCET-08-0125), and Research Fund for the Doctoral Program of Higher Education of China (RFDP-20100071110006). Simulations were carried out at the National High Performance Computing Center of Fudan University. ’ REFERENCES (1) Gray, J. J. Curr. Opin. Struct. Biol. 2004, 14, 110. (2) Mao, S.; Lu, G. H.; Yu, K. H.; Bo, Z.; Chen, J. H. Adv. Mater. 2010, 22, 3521. (3) Zeng, Q. O.; Cheng, J. S.; Tang, L. H.; Liu, X. F.; Liu, Y. Z.; Li, J. H.; Jiang, J. H. Adv. Funct. Mater. 2010, 20, 3366. (4) Toublan, F. J. J.; Boppart, S.; Suslick, K. S. J. Am. Chem. Soc. 2006, 128, 3472. (5) Nel, A. E.; Madler, L.; Velegol, D.; Xia, T.; Hoek, E. M. V.; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M. Nat. Mater. 2009, 8, 543. (6) Bianco, A.; Kostarelos, K.; Prato, M. Curr. Opin. Chem. Biol. 2005, 9, 674. (7) Stefani, M. Int. J. Mol. Sci. 2008, 9, 2515. (8) Stefani, M. Neuroscientist 2007, 13, 519. (9) Dobson, C. M. Nature 2003, 426, 884. (10) Pan, K. M.; Baldwin, M.; Nguyen, J.; Gasset, M.; Serban, A.; Groth, D.; Mehlhorn, I.; Huang, Z. W.; Fletterick, R. J.; Cohen, F. E.; Prusiner, S. B. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 10962. (11) Peretz, D.; Williamson, R. A.; Matsunaga, Y.; Serban, H.; Pinilla, C.; Bastidas, R. B.; Rozenshteyn, R.; James, T. L.; Houghten, R. A.; Cohen, F. E.; Prusiner, S. B.; Burton, D. R. J. Mol. Biol. 1997, 273, 614. (12) Giacomelli, C. E.; Norde, W. Biomacromolecules 2003, 4, 1719. (13) Kowalewski, T.; Holtzman, D. M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3688. (14) Rocha, S.; Krastev, R.; Thunemann, A. F.; Pereira, M. C.; Mohwald, H.; Brezesinski, G. ChemPhysChem 2005, 6, 2527. (15) Ban, T.; Morigaki, K.; Yagi, H.; Kawasaki, T.; Kobayashi, A.; Yuba, S.; Naiki, H.; Goto, Y. J. Biol. Chem. 2006, 281, 33677. (16) Sethuraman, A.; Vedantham, G.; Imoto, T.; Przybycien, T.; Belfort, G. Proteins: Struct., Funct., Bioinf. 2004, 56, 669. 9821

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