lable at ScienceDirect

Biomaterials 29 (2008) 3847–3855

Contents lists avai

Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

Induced stepwise conformational change of human serum albuminon carbon nanotube surfaces

Jia-Wei Shen, Tao Wu*, Qi Wang*, Yu KangDepartment of Chemistry, Zhejiang University, Hangzhou 310027, P. R. China

a r t i c l e i n f o

Article history:Received 7 April 2008Accepted 18 June 2008Available online 9 July 2008

Keywords:Human serum albuminCarbon nanotubeProtein adsorptionStepwise conformational changeProtein-surface interaction

* Corresponding authors. Fax: þ86 571 87951895.E-mail addresses: tao_wu@zju.edu.cn (T. Wu), qiw

0142-9612/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.biomaterials.2008.06.013

a b s t r a c t

Non-covalent adsorption of proteins onto carbon nanotubes is important to understand the environ-mental and biological activity of carbon nanotubes as well as their potential applications in nano-structure fabrication. In this study, the adsorption dynamics and features of a model protein (the Asub-domain of human serum albumin) onto the surfaces of carbon nanotubes with different diameterswere investigated out by molecular dynamics simulation. The adsorption behaviors were observed byboth trajectory and quantitative analyses. During the adsorption process, the secondary structures of a-helices in the model protein were slightly affected. However, the random coils connecting these a-heliceswere strongly affected and this made the tertiary structure of protein change. The conformation andorientation selection of the protein were induced by the properties and the texture of surfaces indicatedby the interaction curve. In addition, the stepwise adsorption dynamics of these processes are found. Themechanism of induced stepwise conformational change of protein on carbon nanotube surfaces wouldbe helpful to better understand the protein–surface interaction at the molecular level.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Since the discovery of carbon nanotubes (CNTs) in 1991 [1],CNTs have raised considerable attention due to their fascinatingstructures and properties (electronic, optical, thermal, mechanicaletc.) [2,3]. Recently, its potential application in biotechnology hasattracted much interest, as CNTs have been reported to exhibitgreat advantages in biosensors [4,5], biomedical devices [6] anddrug delivery systems (DDS) [7,8] etc.

Pristine CNTs are highly hydrophobic, so the main obstacle inthe utilization of CNTs in biological and medicinal chemistry is theirpoor solubility in aqueous-based biological milieu. Biomoleculefunctionalization is one option to overcome such defects. Manyexperimental efforts in this direction have been made through ei-ther covalent or non-covalent interactions between biomoleculesand CNTs. For example, by covalently attaching functional groups tonanotubes, CNTs have been made soluble in different solvents [9–11]. Through such modifications, the water solubility of CNTs isclearly improved and their biocompatibility profile completelytransformed. However, sidewall or tip covalent functionalizationscould alter the electronic structure of the nanotubes by disruptingthe network of sp2-hybridized carbons [12] and/or change thepotential energy surface of the CNT [13]. In addition to covalent

ang@zju.edu.cn (Q. Wang).

All rights reserved.

functionalization, non-covalent bond functionalization is in-creasingly developed as an alternative route to keep the nanotubes’intrinsic mechanical and electrical properties intact. It has beenfound that some proteins could be encapsulated into the innerspace of CNTs [14–16], or non-covalently bound to the sidewalls ofCNTs [17–19]. As for the non-covalent binding of peptides or pro-teins to the sidewall of CNTs, the processes all concern the ad-sorption and/or desorption of these molecules on the CNT surfaces[20–23]. Many experiments with varied methods were performedto qualitatively and quantitatively study the adsorption of peptidesand/or proteins on the CNT surfaces. For example, by using thephage display technique, scanning electron microscopy (SEM) andtransmission electron microscopy (TEM), Wang et al. [20] identifiedpeptides with a selective affinity for CNTs. Their results of con-sensus binding sequences show a motif rich in histidine and tryp-tophan at specific locations, and the binding sequence is flexible,folding into a structure matching the geometry of CNTs. Witus et al.[21] created a series of peptides that individually dispersed CNTswith modifications to coat the nanotube in different environments.The ability of the peptides to individually disperse CNTs withoutaltering their electronic structure is shown by visible and near-IRabsorbance, fluorescence, and regular and vitreous ice cryo-TEM.Karajanagi et al. [22] examined the structure and function of twoenzymes, a-chymotrypsin (CT) and soybean peroxidase (SBP),adsorbed onto carbon nanotubes. FT-IR spectroscopy revealed thatboth enzymes undergo structural changes upon adsorption, withsubstantial secondary structural perturbation observed for CT. The

J.-W. Shen et al. / Biomaterials 29 (2008) 3847–38553848

adsorbed SBP retained up to 30% of its native activity upon ad-sorption, while the adsorbed CT retained only 1%. Atomic forcemicroscopy (AFM) images of the adsorbed enzymes are consistentwith the measurement of enzymatic activity.

These experiments greatly enhanced the understanding of theinteraction between the peptide/protein and the CNT surfaces;however, the atomic details of the interactions taking place at themolecular level are still obscure, due to the limitation of the ex-perimental methods. For example, the details of function, confor-mation and spatial orientation of proteins that are non-covalentlyadsorbed onto the carbon nanotubes cannot be well explored bythese methods. Moreover, the dynamics of the adsorption process,which is fundamental in the biological applications of carbonnanotubes, is poorly understood via these techniques. Neverthe-less, these branches of knowledge are of great importance in un-derstanding the environmental and biological activity of carbonnanotubes as well as their potential applications in nanostructurefabrication. Atomistic molecular simulation presents one of themost direct approaches to investigate the atomic details of theadsorbate–adsorbent surface interactions. It could explore the dy-namics at the molecular level as well as address the effect of surfacechemistry on the adsorption behavior of proteins [24–26]. Localfeatures, including secondary and tertiary structure changes, ori-entation and reorientation of the protein on the interface, charac-teristics of the different amino acid side groups, hydrogen bondsetc. are essential to properly understand protein adsorption onheterogeneous surfaces, and these behaviors can only be accountedfor via atomistic simulations. In addition, it is an excellent methodfor imaging in bionanotechnology, because it could cover the spacescale rather well and cover the time scale up to many nanosecondsfor all-atom molecular simulations. The adsorption and desorptionbehaviors of some proteins on the biomaterials surfaces have beenstudied by atomistic molecular simulations in our group [27–31]. Inthese previous studies, the dominant interaction between proteinsand the inorganic surfaces, the conformation and orientationchanges of model proteins upon adsorption, the dynamic featuresin adsorption and desorption processes etc. were carefully in-vestigated and well understood. All these simulations carried outby our group and others have proved that atomistic molecularsimulation is powerful to explore the protein–surface interaction.Thus it was employed to investigate the adsorption behaviors anddynamics for human serum albumin (HSA) onto the carbon nano-tube surfaces in this study. HSA is the most abundant blood protein,and it is one of the key proteins involved in the biocompatibility forbiomaterials. Hence, in this study, one sub-domain of HSA wasselected as a benchmark system to study the initial adsorptionfeatures and dynamics onto the carbon nanotube surfaces bymolecular dynamics (MD) simulations.

Fig. 1. (a) The model protein was selected from the first three a-helices of the A domain of chare connected by a few amino acids present as regular turns or in a random conformation. T(b) The initial configuration of the model protein and the armchair CNT (16, 16) system beforThe model protein is displayed by a line model combined with the cartoon model. The axial dsections of the CNT are in the x–y plane. In all the simulations, carbon atoms of the CNT wcontaining different diameter CNTs are the same as this one.

2. Simulation method

In this study, all MD simulations were performed by the NAMDprogram [32] with the CHARMM27 force field [33]. The force fieldparameters of carbon nanotubes were obtained from Ref. [34] andall the carbon atoms in carbon nanotubes were set to be neutral.The parameters of Lennard-Jones potential for the cross in-teractions between non-bonded atoms (e.g., CNT–protein, CNT–water) were obtained from the Lorentz–Berthelot combinationrules as in our previous studies [30,31] and others [35,36]. The x-raystructure of chain A of HSA was derived from the protein date bank(ID code 2BXI). Chain A of HSA contains six domains, labeled from Ato F, and these domains form three pairs: A–B, C–D, and E–F. Thereare six a-helices in the A, C, E domains, while B, D and F comprisefour a-helices. In this work, the first three a-helices in the A domainwere selected as the model protein to explore the adsorption be-havior and dynamics of the protein on the surfaces of carbonnanotubes. The helices of the model protein are connected by a fewamino acids present as regular turns or in a random conformation,which is displayed in Fig. 1a by VMD [37], and the model protein isdenoted as ASD (A sub-domain) for convenience in the followingdiscussion. The primary structure of ASD is HKSEV AHRFK DLGEENFKAJ VLIAF AQYLQ QCPFE DHVKL VNEVT EFAKT CVADE SAENC DKand its three-dimensional size is 32.0� 23.7� 38.0 Å3. Single-walled carbon nanotubes with different diameters were in-dividually used in these simulations, namely, armchair (16, 16), (20,20), (24, 24) and (28, 28) carbon nanotubes, with their diametersaround 2.07, 2.59, 3.11 and 3.63 nm, respectively. All atoms in-cluding hydrogen were described explicitly in all the simulations. Atime step of 2 fs was set and a cutoff of non-bonded van der Waalsforce was set with a switching function to start at a distance of 10 Åand to reach zero at 12 Å. Particle mesh Ewald (PME) summationwas used to calculate the long-ranged electrostatic interactions,with a cutoff distance of 12 Å for the separation of the direct andreciprocal space. Periodic boundary conditions were applied for allthe simulations. During these MD simulations, the Langevinmethod was employed to control the constant temperature at 310 Kand the pressure at 101.3 kPa. The axial direction of CNTs was setparallel to the z-direction. Thus the cross sections of the CNTs werein the x–y plane. All carbon atoms of the CNT were fixed; however,proteins were relaxed. These simulations were performed accord-ing to the following steps:

(1) Energy minimization and equilibration of the model protein.The model protein was solvated with SPC water [38] in a waterbox with a box size of 48� 55� 52 Å3, and the system un-derwent a 1000-step energy minimization. Then a MD run ofthe energy-minimized system was carried out for 1 ns until the

ain A of HSA. These helices (marked as A1, A2 and A3, respectively) of the model proteinhe model protein is denoted as ASD (A sub-domain) for convenience in the discussion.e energy minimization and MD simulation. The water molecules are omitted for clarity.irection of the CNT surface was selected to be parallel to the z-direction. Thus the crossere fixed and the protein was relaxed. The initial configurations of the other systems

0.0 0.5 1.0 1.5 2.00.7

0.8

0.9

1.0

d/d

0

Time (ns)

ASD-CNT(16,16)ASD-CNT(20,20)ASD-CNT(24,24)ASD-CNT(28,28)

Fig. 2. The normalized distance of the center-of-mass for the protein to the tube wallof CNT (denoted as CMW distance) for four systems.

J.-W. Shen et al. / Biomaterials 29 (2008) 3847–3855 3849

root mean square deviation (RMSD) and the system’s totalenergy fluctuated around a constant value, which indicatedthat the equilibrium state was reached.

(2) CNTs with different diameters were constructed. Their lengthsare approximately 72 Å, with 30 sections in each CNT. Themodel protein was placed close to the surfaces of CNTs and thedistance of the outmost shell to the surfaces of CNTs is ap-proximately 5 Å to ensure that there is enough space to containseveral layers of water between the proteins and the surfaces.In order to explore the adsorption features of the protein onCNT surfaces with different diameters, one starting orientationof the protein was selected to study the size effect. The modelprotein was rotated to make its cross section in the x–zplane maximum, and it could contact more areas of the sur-faces. The three-dimensional size of the model protein is32.0� 23.7� 38.0 Å after rotation and it was expected that theprotein would get a relatively strong attraction in this startingorientation.

(3) Then the whole system was solvated with SPC water in a waterbox with a box size of 64� y� 86 Å3, where y varied from 118 Åto 131 Å to fit the different diameters of CNTs. In these systems,the number of water molecules contained ranges from 19,409to 20,716, due to the different box sizes of the systems. Fig. 1bshows the initial configuration of the A sub-domain of HSA andthe armchair (16, 16) systems before system minimization andMD simulation. The water molecules are omitted for clarity.The initial configurations of the other systems containing dif-ferent diameter CNTs are similar to this one.

(4) A 10,000-step energy minimization of the whole system(containing the CNT, the model protein and water molecules)was performed at first. Then all of the systems underwent2 ns MD runs until the RMSD and the system’s total energyfluctuated around a constant value. This indicates thata meta-stable state is reached. The main goal of this simu-lation focuses on evaluating the initial adsorption behaviorand the dynamic features, although the protein folding/unfolding events take place over time scales much longerthan several nanoseconds.

It is worth noting that in our MD simulations the SPC watermodel was employed, but the CHARMM27 force field wasdesigned to be used with the TIP3P water model. Actually, boththe SPC and the TIP3P water models are three-site models andthe differences are only in the parameters rO–H (O–H bondlength), qHOH (H–O–H angle) and atom charges. It was reportedthat the SPC model gives a reasonable description for the struc-tural and thermodynamic properties of liquid water when it iscompared to the experimental data [39]; especially, the dielectricproperties obtained by the SPC model are in excellent agreementwith the data available in the literature [40]. Therefore, the SPCmodel is also recommended for molecular simulations of aqueoussolution for biomolecules [38]. For the semi-quantitative study ofprotein adsorption in force field-based MD simulation, the dif-ferences between these two water models were supposed to havea weak effect on the eventual results. Therefore, in our previousstudies concerning protein adsorption and desorption on nano-material surfaces [27–31], CHARMM27/SPC parameterizationachieved reasonable results and they are consistent with manybiological data. The use of CHARMM27/SPC parameters in thisstudy could also explore different adsorption behaviors of proteinon the hydrophilic and hydrophobic surfaces of nanomaterialsbased on these studies. However, there is no literature availablereporting the effect of water models on protein adsorption ontothe nanomaterial surfaces and the selection of the water modelmay influence the adsorption behavior in our work. Thus, studiesare needed to further validate the calculation accuracy for protein

adsorption. The time-dependent interaction, Eint(t), for all thesystems in MD simulations are defined as follows:

EintðtÞ ¼ ECNTþHSAðtÞ�ECNTðtÞ�EHSAðtÞ (1)

In Eq. (1), Eint(t) stands for the total interaction between the modelprotein and the CNT surfaces at time t during the MD simulation,and ECNT þ HSA(t), ECNT(t) and EHSA(t) are the total potential energy ofthe CNT–protein complex, the potential energy of CNT and that ofthe protein at time t during MD simulations, respectively. Thesedata were derived from the preserved frames in the simulations,and the water molecules were removed in all frames prior to theanalysis for simplicity. However, these data have already taken theeffect of water into account, because Eq. (1) is based on the pre-served frames of simulations in the water solvation condition. It isalso worth noting that Eint(t) is not a direct calculation of the in-teraction energy between the protein and the nanotubes but aninstantaneous measurement of cross interaction between theprotein and the nanotubes in the simulations. Actually, it was usedas a quantitative indicator for the interaction energy, and we con-sider that it could provide us more information on the in-stantaneous interaction between the protein and the nanotubes.This method has been successfully applied to investigate the ad-sorption and desorption of proteins in inorganic material surfaces[29–31].

3. Results and discussion

3.1. Effective adsorption

In MD simulations, the adsorption phenomena were observed inall the systems and reflected by the trajectory animation of everysimulation. The model protein, the A sub-domain of HSA, smoothlymoved closer to the tube walls of CNTs in all the systems accom-plished with a local rearrangement of the protein approaching theinterface. Fig. 2 shows the normalized distance of the center-of-mass for the protein to the tube wall (denoted as the CMW distancehenceforth), d/d0, in all the MD simulations. Here the variabled represents the distance of the center-of-mass for the protein tothe tube wall of CNTs, and d0 is the initial value of d before MD runs.Except for the ASD-CNT (24, 24) system, it is visible that the CMWdistances gradually decrease, although there is some fluctuation ford/d0 during the adsorption process, which corresponds to the sur-face induced conformational change of protein or the rearrange-ment of the residues near the CNT surfaces. The final value of the

J.-W. Shen et al. / Biomaterials 29 (2008) 3847–38553850

J.-W. Shen et al. / Biomaterials 29 (2008) 3847–3855 3851

CMW distances of the ASD-CNT (16, 16), CNT (20, 20) and CNT (28,28) systems is 0.821, 0.842 and 0.769, which imply that the center-of-mass of the A sub-domain moves much closer to the tube wall ofthe CNT. The decrease of the CMW distance of these three systemsqualitatively indicates that the adsorption of protein ASD to thesurfaces of CNTs occurred. It is also notable that the CMW distanceof the ASD-CNT (24, 24) system sharply fluctuates and reaches0.965 after the 2 ns MD run. It was observed in the trajectory thatthe C-terminus of this protein moves randomly and translates awayfrom the surfaces in the last 600 ps. Thus, the CMW distance of thissystem does not exhibit the same trend as the others. However, theresidues in the vicinity of the surfaces of CNT (24,24) also undergoobvious rearrangements due to the attraction of the surfaces andsome of the residues are adsorbed onto the surfaces. The adsorptionof some residues could still be observed and this is addressed in thefollowing discussions. The adsorption behavior of protein HSA ontothe CNT-coated silica (Si/SiO2) surfaces has been observed by anexperiment [41]. In addition to the HSA adsorption onto the CNTsurfaces, bovine serum albumin (BSA) was found to form solublestable complexes with carbonaceous nanomaterials [42,43], whichis consistent with our simulations.

3.2. Conformation and orientation of adsorbed residues

Fig. 3 shows the final conformations of the adsorbed protein andthe residues adsorbed in four systems after 2 ns MD simulations. Inthese snapshots, the water molecules are omitted for clarity. Itcould be obviously observed from the side view that the atoms nearthe tube wall of the CNTs rearranged to fit the curving surfaces ofthe CNTs, though the final orientation and conformation of theprotein are different as the curvature of the CNT varies. It indicatesthe effective attraction of the surfaces to the protein. In Raffaini’ssimulations [25,44] concerning the interaction between modelproteins and graphite, the interaction energy was plotted asa function of the number of residues being less than 5 Å from thesurfaces and this function was well fitted. Though in their simula-tions the proteins were adsorbed onto the graphite surfaces whilein our simulations the model protein was adsorbed to the CNTsurfaces, the distance is still a reasonable value to qualitativelyestimate the interaction strength between the protein and thesurfaces of the CNT, in other words, to qualitatively judge whetherthe residues were adsorbed to the surfaces. In the top view of themodel protein adsorbing to the CNTs in Fig. 3, the whole protein isdisplayed by a transparent cartoon model but only the atoms of theprotein within 5 Å around the CNT tube walls (less than 5 Å fromthe surfaces) are displayed by the licorice model for clarity. As forthe system ASD-CNT (16, 16), the adsorbed residues are Val38,Lys39, Asn42, Glu43 and Glu46. The side chain in the Val residue isisopropyl, which is a non-polar group; thus, it is comprehensiblethat Val38 could be easily adsorbed to the surfaces due to the vander Waals interaction as well as the hydrophobic interaction. Inaddition, it is also found that the hydrogen atoms in the methylgroup in the side chain in Val38 tended to be exposed to the sur-faces of the CNT, and it is inferred that this orientation is morefavorable due to the hydrophobic interaction. Because in our model,the carbon atoms of CNT have no net charge, the polar and chargedresidue could also adsorb to the surfaces due to the van der Waalsattractive interaction. In the adsorbed Lys39, Glu43 and Glu46, theorientations of alkylene [–(CH2)n–] in their side chain were almost

Fig. 3. Side view of (a1) ASD-CNT (16, 16), (b1) ASD-CNT (20, 20), (c1) ASD-CNT (24, 24), (d120), (c2) ASD-CNT (24, 24), (d2) ASD-CNT (28, 28) systems after MD simulations. The snapshothe cartoon model, and the CNT is displayed by a VDW model. The proteins in the top view aCNT tube wall are displayed by the licorice model for clarity. The color in the cartoon model iresidues (gray) and polar residues (green). Water molecules are omitted for clarity. In (a2),

parallel to the surfaces of CNTs. In addition, the hydrogen atoms inalkylene, but not the functional group in these side chains, areexposed to the surfaces of the CNT. Many of the functional groups inthe side chains of the adsorbed residues, like –NH3

þ and –COO�,were not included if they are not located within 5 Å around the tubewall. A similar phenomenon was also observed in Asn42. In manyprevious reports [15,35,45], it was revealed that the van der Waalsinteraction between biomacromolecules and carbon nanotube isthe dominant factor and the carbon atoms of CNT were set to beneutral in these studies. Hence we considered that this approxi-mation could be valuable for achieving reasonable and semi-quantitative conclusions. The residue types and orientations in theother three systems are similar to that of the system ASD-CNT (16,16); however, the number of adsorbed residues in the ASD-CNT (20,20) and ASD-CNT (28, 28) systems is a little more than that in ASD-CNT (16, 16). Moreover, it was found that the Phe residues wereadsorbed in these two systems, though they are not identical:Phe34 in ASD-CNT (20, 20) and Phe47 in ASD-CNT (28,28). In thefinal state, the phenyl rings in both systems adopt orientations thatare parallel to the surface. In this orientation, the contact area of thephenyl ring to the surfaces is the maximum, and thus it achievesthe maximum interaction with the CNT [46]. In addition to the vander Waals attraction and hydrophobic interaction between thephenyl ring and the surfaces, the p–p stacking is also a key factorthat drives phenyl to this orientation [47,48]. All the residues thatwere adsorbed in the four systems in meta-stable states are sum-marized in Table 1.

3.3. Dynamics of protein adsorption

In order to further investigate the dynamics of the adsorptionprocess, the interactions between the model protein and CNTs asa function of the simulation time were plotted in Fig. 4. All theenergy curves follow a decreasing trend, which implies the effec-tive interaction between the protein and the surfaces. As illustratedabove, the atoms in the neighborhood of the tube wall of CNTsrearranged to fit the curving surfaces of CNTs; however, this processis stepwise. From the observation of Eint changes and the trajectoryanimation, adsorption always accompanies conformational rear-rangement and reorientation of the residues that are close to thesurfaces; so the decrease of interaction is not linear. Instead, theprotein may overcome an energy barrier after conformationalchange, and then some residues move gradually closer to the sur-faces and were finally kept within approximately 5 Å from the tubewall. Moreover, in the movement of these residues, they tend toadopt favorable orientations. For example, the relatively longalkylene in Lys leans to spread its side chain on the CNT surfacesand make the hydrogen atoms in alkylene point to the surface.Generally, phenyl in Phe tends to make the plane of the ring parallelto the surface. All of these dynamic features are for the purpose thatmakes the whole protein possess the maximum interaction withthe surface and stabilize the adsorption of the protein onto thesurfaces. Fig. 4 reflects these features, in which the interactioncurves decrease sharply in a relatively short time period, though itfluctuates around a specific value most of the time. For instance, theinteraction in the ASD-CNT (20, 20) system decreases from�38.6 kcal mol�1 to �60.0 kcal mol�1 between 0.72 ns and 0.93 ns,which could be mainly attributed to the adsorption of Asn47 andVal38. Similarly, from 1.46 ns to 1.54 ns, the interaction in the ASD-

) ASD-CNT (28, 28) systems, and top view of (a2) ASD-CNT (16, 16), (b2) ASD-CNT (20,ts of the model protein in the side view are displayed by the line model combined withre displayed by the cartoon model and only the atoms of protein within 5 Å around then all the snapshots represents the basic residues (blue), acidic residues (red), non-polar(b2), (c2) and (d2), the nanotubes were partially displayed to give the best view.

Table 1The adsorbed residues and the interaction between the model protein and CNT inthe meta-stable state in four systems

System Residues adsorbed Interaction inmeta-stablestate (kcal mol�1)

ASD-CNT (16, 16) Val38 Lys39 Asn42 Glu43 Glu46 �58.5ASD-CNT (20, 20) Phe34 Glu35 Val38 Lys39 Asn42 Glu46 �84.0ASD-CNT (24, 24) Glu35 Val38 Lys39 Asn42 �63.6ASD-CNT (28, 28) Lys39 Asn42 Glu43 Glu46 Phe47 Thr50 �74.7

J.-W. Shen et al. / Biomaterials 29 (2008) 3847–38553852

CNT (20, 20) system decreases from �71.4 kcal mol�1 to�84.9 kcal mol�1, and this 12.5 kcal mol�1 energy drop is mainlybecause Phe34 is adsorbed and takes a favorable orientation in theadsorbed state. This value is larger than the comparable firstprinciple calculation [48], but in this time period, other parts of theprotein still exhibit the trend to move closer to the surfaces; so thereduction of interaction caused by the adsorption of Phe34 is stillreasonable. As discussed above, several steps exist in the in-teraction curve, which implies that the protein could adjust itsconformation and orientation to fit the arrangement of carbonatoms in the surfaces of CNTs; or in other words, the conformationand orientation selection of the protein are induced by the prop-erties and the texture of the surfaces. After overcoming the energybarrier caused by the conformational rearrangement and/or reor-ientation, one or more residues which have an affinity to the sur-faces could be readily adsorbed and make the interaction curveprogress to the next step and finally achieve a relatively stable state.In a recent paper of Matsuura et al. [42], they inferred that therearrangements of the amino acids and the reconstruction of thehydrophobic packing during denaturation and refolding processesinfluence the protein selectivity of the SWNT dispersion. The

0.0 0.5 1.0 1.5 2.0-80

-60

-40

-20

0

In

teractio

n (kcal/m

ol)

Time (ns)

a

c

0.0 0.5 1.0 1.5 2.0Time (ns)

-80

-60

-40

-20

In

teractio

n (kcal/m

ol)

Fig. 4. Interactions between the model protein and CNT as a function of the simulation time i28) systems.

dynamics of stepwise rearrangements of the residues in our sim-ulation consists with this result and provides more details at theatomic level. This adsorption dynamics seems somewhat differentfrom the desorption behavior of some other proteins to hydrophilicsurfaces reported in our previous papers [28,30]. In these studies,the surface texture of hydroxyapatite is very different from that ofCNTs, and the dominant interactions taking place at the interfaceare the electrostatic interaction and water-bridged H-bond, whilein this study, the main interactions are attributed to the van derWaals and hydrophobic interactions. All these investigations revealthat the dynamics of protein adsorption and desorption would becontrolled by the property and the texture of material surfaces.Thus, it is possible to regulate the adsorption and/or desorptionbehaviors via surface design and modification.

The interactions of the four systems in meta-stable states aresummarized in Table 1 by averaging the data for the last 200 ps. It isfound that the systems that contain the Phe residue adsorbedpossess a larger interaction. It agrees with the experimental resultthat peptides containing aromatic rings have a higher affinity withCNTs [20,49]. Another finding is that except for the ASD-CNT (20,20) system, the interaction in the meta-stable state increases withthe increase in diameter. In a recent study concerning the dynamicsof the encapsulation of a collagen-like peptide into carbon nano-tubes in our group, the insertion is diameter selective. Hence, theinteraction and the CNT diameter are correlative. In this situation,besides the adsorption of Phe, the relatively large interaction in theASD-CNT (20, 20) system may be a result of the fitness of theprotein conformation to the specific curvature of the CNT surfaces.This phenomenon, that the polymer-surface or protein-surfaceinteractions were not always monotonic functions of the curvatureof inorganic material surfaces [46,50], was also found in othersimulations and the experiments.

b

d

0.0 0.5 1.0 1.5 2.0Time (ns)

0.0 0.5 1.0 1.5 2.0Time (ns)

-100

-80

-60

-40

-20

0

-100

-80

-60

-40

-20

0

In

teractio

n (kcal/m

ol)

In

tera

ctio

n (kcal/m

ol)

n (a) ASD-CNT (16, 16), (b) ASD-CNT (20, 20), (c) ASD-CNT (24, 24) and (d) ASD-CNT (28,

0 2 4 6 8 10 120

100

200

300

400

500ASD-CNT (16, 16)ASD-CNT (20, 20)ASD-CNT (24, 24)ASD-CNT (28, 28)

Nu

mb

er o

f a

to

ms

Distance to tube wall (Angstrom)

2 4 6 8 10 12Distance to tube wall (Angstrom)

-10

-5

0

ASD-CNT (16, 16)ASD-CNT (20, 20)ASD-CNT (24, 24)ASD-CNT (28, 28)

In

te

ra

ctio

n / N

oA

(k

ca

l/m

ol)

ba

Fig. 5. (a) Number of atoms (NoA) in the protein within a certain distance from the tube wall (less than a certain distance from the curved surface) in the four systems after MDsimulations. (b) Interactions in the meta-stable state divided by NoA within a certain distance from the tube wall in the four systems.

J.-W. Shen et al. / Biomaterials 29 (2008) 3847–3855 3853

To explore the relationship between the interaction strength inthe meta-stable state and the number of atoms adsorbed in theprotein under the water solvated condition, the number of atoms inthe protein within a certain distance from the tube wall (denoted asNoA in the following discussion) was plotted in Fig. 5a. In all thesystems, the atoms appeared around 2.5 Å. As the distance in-creases, the NoA values of all the systems increase gradually. Thesequence of NoA values is approximately ASD-CNT (20, 20)>ASD-CNT (28, 28)>ASD-CNT (24, 24)>ASD-CNT (16, 16) under 6 Å,which coincides with the strength of the interaction, thougharound 5 Å the NoA value in ASD-CNT (16, 16) is slightly larger thanthat in ASD-CNT (24, 24). As the interaction strength is basically

0

1

2

3

RM

SD

(A

ng

stro

m)

a

c

0.0 0.5 1.0 1.5 2.0Time (ns)

0.0 0.5 1.0 1.5 2.0Time (ns)

0

1

2

3

4

5

RM

SD

(A

ng

stro

m)

ASDA1A2A3

ASDA1A2A3

Fig. 6. Root mean square deviation (RMSD) of the A sub-domain, and its individual a-heliceCNT (24,24) and (d) ASD-CNT (28, 28) systems.

determined by the attraction between the surfaces and the adja-cent atoms, the interactions between the protein and the surfacesin the meta-stable state in the four systems were divided by NoAwithin a certain distance from the tube wall, as shown in Fig. 5b. Itis obvious that the divided interaction sharply increases from 3 Å to6 Å in all systems, and gradually becomes a platform after 6 Å witha small difference among these systems. So it is considered that theatoms within 6 Å from the tube wall mainly determine the attrac-tive strength of CNT surfaces to the proteins, and 6 Å could beregarded as the effective interaction distance. The exact values ofinteraction divided by NoA within 6 Å from the tube wall are 0.77,0.72, 0.72 and 0.78 kcal mol�1 in ASD-CNT (16, 16), ASD-CNT (20,

0.0 0.5 1.0 1.5 2.00

1

2

3

4

5b

d

RM

SD

(A

ng

stro

m)

Time (ns)

0.0 0.5 1.0 1.5 2.0Time (ns)

ASDA1A2A3

ASDA1A2A3

0

1

2

3

4

5

RM

SD

(A

ng

stro

m)

s A1, A2, A3 in 2 ns simulations for (a) ASD-CNT (16, 16), (b) ASD-CNT (20, 20), (c) ASD-

J.-W. Shen et al. / Biomaterials 29 (2008) 3847–38553854

20), ASD-CNT (24,24) and ASD-CNT (28, 28) systems, respectively.Thus the averaged values (0.77, 0.72, 0.72 and 0.78 kcal mol�1) andthe number of atoms within 6 Å from the CNT surfaces may be usedto qualitatively evaluate the interaction strength between theproteins and the CNT surfaces in the water solvated condition bysimple multiplication.

3.4. Structure deformation of protein

Fig. 6 displays the root mean square deviation (RMSD) of thebackbone of the model protein, and individual a-helices A1, A2, A3in all the simulations. It is obvious that the conformation change ofall a-helices is slight, especially for a-helices A1 and A2 (within 1 Åmost of the time). The RMSD of the A3 helix is a little higher thanthe others because this helix is in the nearest region of the surfacescompared with the others. This slight structure change of a-helicesprobably originated from the partial unfolding of the a-helices inalbumin [42]. However, the total RMSD value for the whole proteinin its final state is much more than those a-helices except for theASD-CNT (16, 16) system. This indicates that the coils that connectthese a-helices undergo a relatively large deformation upon theadsorption of protein. The tertiary structure of the model proteinmay change a lot basically owing to the large deformation of thecoils although the secondary structure of the protein is slightlyaffected upon adsorption, which can be deduced from the confor-mational change of these a-helices. For example, at 1.4 ns in theASD-CNT (24, 24) system, the large RMSD change for the wholeprotein basically originates from the conformational change of thecoils while the RMSD for the other three a-helices is within 1 Å.After 1.4 ns, the coils at the C-terminus are randomly moving andtranslating away from the surfaces that make the RMSD of A2 andA3 helices rise. Moreover, this movement causes the CMW distanceof the ASD-CNT (24, 24) system to increase as shown in Fig. 2, al-though the protein is still in the adsorbed state. In the research ofDutta et al. [51], albumin was identified as the most abundantprotein adsorbed from FBS and human serum/plasma on CNTs. It iswell established that the liver is highly efficient at clearingdamaged albumin from the circulation because damaged orstructurally altered albumin is a well-known ligand for scavengerreceptors. Therefore, the albumin-carbon nanotube complex andthe associated structural changes in the adsorbed albumin are re-sponsible for targeting carbonaceous nanomaterials to scavengerreceptor uptake in their study. Their result indicates that albuminundergoes structure deformation when it is adsorbed onto CNTsurfaces, and this is consistent with our findings by simulations. Inconclusion, the adsorption of protein onto the surfaces of CNTprobably changes its tertiary structure. In addition, part of itsactivity may be lose due to structure deformation, which wasobserved by the experiments [22].

Although our simulations achieved reasonable results, there arestill some limitations in these simulations, which may be the ob-stacle to better understand the protein adsorption features anddynamics on nanomaterial surfaces. First, these results are fora specific starting orientation of the protein and different startingorientations are likely to result in different adsorption behaviors.Secondly, 2 ns MD simulation could achieve the meta-stable statebut it is probably not long enough for the system to reach a trueequilibrium state and longer simulation times are needed to fullyunderstand various system behaviors. Finally, these results arebased on the CHARMM27/SPC parameterization, which has notbeen validated for this type of adsorption system, and furtherstudies are needed to validate the accuracy of the predicted results.Our future studies would make an effort to overcome these limi-tations and better understand the protein–nanomaterialsinteraction.

4. Conclusion

The adsorption behaviors and features of a model protein, the Asub-domain of human serum albumin, on the surfaces of carbonnanotubes with different diameters were investigated out bymolecular dynamics simulations. The normalized distances of thecenter-of-mass for the protein to the wall of the carbon nanotubesand the interactions between the protein and the surfaces confirmthe effective attraction and protein adsorption. In addition to thenon-polar residues like Val and Phe, some polar and charged resi-dues that contain the alkylene chain segment [–(CH2)n–] were alsoobserved to be adsorbed in specific orientations. The conformationand orientation selection of the protein were induced by theproperties and the texture of surfaces, which is deduced fromthe interaction curve. After overcoming various energy barriers ofthe system, one or more residues in the protein which have anaffinity to the surface could be readily adsorbed and drive the in-teraction curve to the next step. This stepwise adsorption dynamicsseems somewhat different from the desorption behavior of someother proteins to a hydrophilic surface revealed in our earlier study.In addition, the interactions in the meta-stable state in all thesystems indicate that the system which contains adsorbed aromaticrings possesses a stronger interaction between the protein and CNTsurfaces, and has a higher affinity to the CNTs. In addition, a simplequalitative estimation of the interaction strength between theproteins and CNTs was performed. During the adsorption process,the secondary structure of a-helices in the model protein wasslightly affected. However, the random coils connecting these a-helices were strongly affected and this might alter the tertiarystructure of the protein. These conformational changes may highlyaffect the activity of the protein. Although there are still somelimitations in our simulations, this work achieved reasonable re-sults and is consistent with some of the experimental results. Ourwork could be helpful to understand protein–surface interactionsat the molecular level and guide non-covalent bond functionali-zation of carbon nanotubes; thus these findings may be useful forresearch and applications in the fields of biomaterials, biosensors,biomedical devices, and drug delivery.

Acknowledgements

The financial support of the National Natural Science Founda-tion of China (grant nos. 60533050 and 20503025) and of theZhejiang Provincial Natural Science Foundation of China (grant no.R407042) is gratefully acknowledged.

References

[1] Iijima S. Helical microtubules of graphitic carbon. Nature 1991;354:56–8.[2] Guldi DM, Rahman GMA, Sgobba V, Ehli C. Multifunctional molecular carbon

materials – from fullerenes to carbon nanotubes. Chem Soc Rev 2006;35:471–87.

[3] Goldberger J, Fan R, Yang PD. Inorganic nanotubes: a novel platform fornanofluidics. Acc Chem Res 2006;39:239–48.

[4] Chen RJ, Bangsaruntip S, Drouvalakis KA, Kam NWS, Shim M, Li YM, et al.Noncovalent functionalization of carbon nanotubes for highly specific elec-tronic biosensors. Proc Natl Acad Sci U S A 2003;100:4984–9.

[5] Zhang Y-B, Kanungo M, Ho AJ, Freimuth P, van der Lelie D, Chen M, et al.Functionalized carbon nanotubes for detecting viral proteins. Nano Lett 2007;7:3086–91.

[6] Baughman RH, Cui CX, Zakhidov AA, Iqbal Z, Barisci JN, Spinks GM, et al.Carbon nanotube actuators. Science 1999;284:1340–4.

[7] Kam NWS, Jessop TC, Wender PA, Dai H. Nanotube molecular transporters:internalization of carbon nanotube-protein conjugates into mammalian cells. JAm Chem Soc 2004;126:6850–1.

[8] Kam NWS, Liu Z, Dai H. Functionalization of carbon nanotubes via cleavabledisulfide bonds for efficient intracellular delivery of siRNA and potent genesilencing. J Am Chem Soc 2005;127:12492–3.

[9] Zhao W, Song C, Pehrsson PE. Water-soluble and optically pH-sensitive single-walled carbon nanotubes from surface modification. J Am Chem Soc 2002;124:12418–9.

J.-W. Shen et al. / Biomaterials 29 (2008) 3847–3855 3855

[10] Hirsch A. Functionalization of single-walled carbon nanotubes. Angew ChemInt Ed Engl 2002;41:1853–9.

[11] Georgakilas V, Kordatos K, Prato M, Guldi DM, Holzinger M, Hirsch A. Organicfunctionalization of carbon nanotubes. J Am Chem Soc 2002;124:760–1.

[12] Boul PJ, Liu J, Mickelson ET, Huffman CB, Ericson LM, Chiang IW, et al. Re-versible sidewall functionalization of buckytubes. Chem Phys Lett 1999;310:367–72.

[13] Liu YC, Shen JW, Gubbins KE, Moore JD, Wu T, Wang Q. Diffusion dynamicsof water controlled by topology of potential energy surface inside carbonnanotubes. Phys Rev B 2008;77:125438.

[14] Liu YC, Wang Q. Dynamic behaviors on zadaxin getting into carbon nanotubes.J Chem Phys 2007;126:124901.

[15] Kang Y, Wang Q, Liu YC, Wu T, Chen Q, Guan WJ. Dynamic mechanism ofcollagen-like peptide encapsulated into carbon nanotubes. J Phys Chem B2008;112:4801–7.

[16] Davis JJ, Green MLH, Hill HAO, Leung YC, Sadler PJ, Sloan J, et al. Theimmobilisation of proteins in carbon nanotubes. Inorg Chim Acta 1997;272:261–6.

[17] Karajanagi SS, Yang H, Asuri P, Sellitto E, Dordick JS, Kane RS. Protein-assisted solubilization of single-walled carbon nanotubes. Langmuir 2006;22:1392–5.

[18] Kurppa K, Jiang H, Szilvay GR, Nasibulin AG, Kauppinen EI, Linder MB. Con-trolled hybrid nanostructures through protein-mediated noncovalent func-tionalization of carbon nanotubes. Angew Chem Int Ed Engl 2007;46:6446–9.

[19] Kam NWS, Dai H. Carbon nanotubes as intracellular protein transporters:generality and biological functionality. J Am Chem Soc 2005;127:6021–6.

[20] Wang SQ, Humphreys ES, Chung S-Y, Delduco DF, Lustig SR, Wang H, et al.Peptide with selective affinity for carbon nanotubes. Nat Mater 2003;2:196–200.

[21] Witus LS, Rocha J-DR, Yuwono VM, Paramonov SE, Weisman RB, Hartgerink JD.Peptides that non-covalently functionalize single-walled carbon nanotubes togive controlled solubility characteristics. J Mater Chem 2007;17:1909–15.

[22] Karajanagi SS, Vertegel AA, Kane RS, Dordick JS. Structure and function ofenzymes adsorbed onto single-walled carbon nanotubes. Langmuir 2004;20:11594–9.

[23] Su ZD, Leung T, Honek JF. Conformational selectivity of peptides for single-walled carbon nanotubes. J Phys Chem B 2006;110:23623–7.

[24] Agashe M, Raut V, Stuart SJ, Latour RA. Molecular simulation to characterizethe adsorption behavior of a fibrinogen g-chain fragment. Langmuir 2005;21:1103–17.

[25] Raffaini G, Ganazzoli F. Surface ordering of proteins adsorbed on graphite. JPhys Chem B 2004;108:13850–4.

[26] Cormack AN, Lewis RJ, Goldstein AH. Computer simulation of protein ad-sorption to a material surface in aqueous solution: biomaterials modeling ofa ternary system. J Phys Chem B 2004;108:20408–18.

[27] Chen X, Wang Q, Shen JW, Pan HH, Wu T. Adsorption of leucine-rich amelo-genin protein on hydroxyapatite (001) surface through –COO� claws. J PhysChem C 2007;111:1284–90.

[28] Dong XL, Wang Q, Wu T, Pan HH. Understanding adsorption-desorptiondynamics of BMP-2 on hydroxyapatite (001) surface. Biophys J 2007;93:750–9.

[29] Zhou HL, Wu T, Dong XL, Wang Q, Shen JW. Adsorption mechanism of BMP-7on hydroxyapatite (001) surfaces. Biochem Biophys Res Commun 2007;361:91–6.

[30] Shen JW, Wu T, Wang Q, Pan HH. Molecular simulation of protein adsorptionand desorption on hydroxyapatite surfaces. Biomaterials 2008;29:513–32.

[31] Chen X, Wu T, Wang Q, Shen JW. Shield effect of silicate on adsorption ofprotein onto silicon-doped hydroxyapatite (100) surface. Biomaterials 2008;29:2423–32.

[32] Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, Villa E, et al. Scalablemolecular dynamics with NAMD. J Comput Chem 2005;26:1781–802.

[33] MacKerell Jr AD, Bashford D, Bellott M, Dunbrack Jr RL, Evanseck JD, Field MJ,et al. All-atom empirical potential for molecular modeling and dynamicsstudies of proteins. J Phys Chem B 1998;102:3586–616.

[34] Walther JH, Jaffe R, Halicioglu T, Koumoutsakos P. Carbon nanotubes in water:structural characteristics and energetics. J Phys Chem B 2001;105:9980–7.

[35] Gao HJ, Kong Y. Simulation of DNA–nanotube interactions. Annu Rev MaterRes 2004;34:123–50.

[36] Ravichandran S, Madura JD, Talbot J. A Brownian dynamics study of the initialstages of hen egg-white lysozyme adsorption at a solid interface. J Phys ChemB 2001;105:3610–3.

[37] Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics. J MolGraphics 1996;14:33–8.

[38] Zielkiewicz J. Structural properties of water: comparison of the SPC, SPCE,TIP4P, and TIP5P models of water. J Chem Phys 2005;123:104501.

[39] Jorgensen WL, Chandrasekhar J, Madura JD. Comparison of simple potentialfunctions for simulating liquid water. J Chem Phys 1983;79:926–35.

[40] Hochtl P, Boresch S, Bitomsky W, Steinhauser O. Rationalization of the di-electric properties of common three-site water models in terms of their forcefield parameters. J Chem Phys 1998;109:4927–37.

[41] Valenti LE, Fiorito PA, Garcıa CD, Giacomelli CE. The adsorption-desorptionprocess of bovine serum albumin on carbon nanotubes. J Colloid Interface Sci2007;307:349–56.

[42] Matsuura K, Saito T, Okazaki T, Ohshima S, Yumura M, Iijima S. Selectivity ofwater-soluble proteins in single-walled carbon nanotube dispersions. ChemPhys Lett 2006;429:497–502.

[43] Belgorodsky B, Fadeev L, Kolsenik J, Gozin M. Formation of a soluble stablecomplex between pristine C60-fullerene and a native blood protein. Chem-BioChem 2006;7:1783–9.

[44] Raffaini G, Ganazzoli F. Molecular dynamics simulation of the adsorption ofa fibronectin module on a graphite surface. Langmuir 2004;20:3371–8.

[45] Gao HJ, Kong Y, Cui DX. Spontaneous insertion of DNA oligonucleotides intocarbon nanotubes. Nano Lett 2003;3:471–3.

[46] Yang MJ, Koutsos V, Zaiser M. Interactions between polymers and carbonnanotubes: a molecular dynamics study. J Phys Chem B 2005;109:10009–14.

[47] Snow ES, Perkins FK, Houser EJ, Badescu SC, Reinecke TL. Chemical detectionwith a single-walled carbon nanotube capacitor. Science 2005;307:1942–5.

[48] Woods LM, Badescu SC, Reinecke TL. Adsorption of simple benzene derivativeson carbon nanotubes. Phys Rev B 2007;75:155415.

[49] Chen RJ, Zhan YG, Wang DW, Dai HJ. Noncovalent sidewall functionalization ofsingle-walled carbon nanotubes for protein immobilization. J Am Chem Soc2001;123:3838–9.

[50] Roach P, Farrar D, Perry CC. Surface tailoring for controlled protein adsorption:effect of topography at the nanometer scale and chemistry. J Am Chem Soc2006;128:3939–45.

[51] Dutta D, Sundaram SK, Teeguarden JG, Joseph B, Fifield LS, Jacobs JM. Adsorbedproteins influence the biological activity and molecular targeting of nano-materials. Toxicol Sci 2007;100:303–15.