A molecular modeling study of the effect of surfacechemistry on the adsorption of a fibronectin fragmentspanning the 7–10th type III repeats

Kerry Wilson,1 Steven J. Stuart,2 Andres Garcia,3 Robert A. Latour, Jr.11Department of Bioengineering, Clemson University, 501 Rhodes Engineering Research Building, Clemson,South Carolina 296342Department of Chemistry, Clemson University, Hunter Laboratories, P.O. Box 340973, Clemson,South Carolina 296343Woodruff School of Mechanical Engineering, Georgia Institute of Technology, 315 Ferst Drive, Room 2314 IBB,Atlanta, Georgia 30332

Received 22 August 2003; revised 5 February 2004; accepted 10 February 2004Published online 3 May 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30042

Abstract: Although it is well documented that proteinsadsorb onto biomaterial surfaces, relatively little is quanti-tatively understood about the effects of adsorption on pro-tein orientation and conformation. Because this is the pri-mary determining factor of protein bioactivity, the ability toaccurately predict a protein’s orientation and conformationfollowing adsorption will be essential for the rational designof biomaterial surfaces to control biological responses. Forcefield-based computational chemistry methods provide anexcellent means to theoretically address this issue, with thenontrivial requirement that the force field must be tailoredto appropriately represent protein adsorption behavior. Ac-cordingly, we have modified an existing force field(CHARMm) based on semiempirical quantum-mechanicalpeptide adsorption data to enable it to simulate protein

adsorption behavior in an implicit aqueous environment.This modified force field was then applied to predict theadsorption behavior of the 7–10 type III repeats of fibronec-tin on functionalized surfaces. Predicted changes in adsorp-tion energy and adsorption-induced conformation as a func-tion of surface chemistry were found to correlate well withexperimentally observed trends for these same systems. Thiswork represents a first attempt towards the development ofa molecular mechanics force field that is specifically param-eterized to accurately simulate protein adsorption to bioma-terial surfaces. © 2004 Wiley Periodicals, Inc. J Biomed MaterRes 69A: 686–698, 2004

Key words: protein adsorption; fibronectin; molecular mod-eling; self-assembled monolayers; biomaterials

INTRODUCTION

Cell adhesion to synthetic substrates is critical in thedesign of biomedical and biotechnological devices. Ofthe many factors that influence cell adhesion, the pres-ence of a biologically active adsorbed protein layer hasbeen found to be one of the most important.1–4 Whenan artificial material comes into contact with any bio-logical fluid, proteins such as albumin, immunoglob-ulin, vitronectin, fibrinogen, and fibronectin will accu-mulate at the material surface. This adsorption processcan cause a protein molecule to undergo conforma-tional changes that denature epitopes for the binding

of cellular adhesion receptors, such as integrins,4 orperhaps expose other domains that may provide sig-nals to inflammatory cells, such as macrophages.5

Thus, it would be advantageous to be able to quanti-tatively predict the orientational and conformationalrearrangements that occur due to adsorption to syn-thetic surfaces as an approach to proactively designsurfaces to direct cellular response.

Many experimental methods exist that provide in-formation about adsorbed proteins, such as ellipsom-etry, cell adhesion assays, surface plasmon resonancespectroscopy (SPR), and enzyme-linked immunosor-bent assay (ELISA).4,6 Recently, Garcia et al. con-ducted SPR, radiolabeling, and ELISA studies to char-acterize the initial association rate and amount ofadsorption of fibronectin (Fn) and a fragment contain-ing the 7–10 type III repeats of fibronectin (FnIII7–10)onto functionalized gold-alkanethiol self-assembledmonolayer (SAM) surfaces.4,6 The surface functional-

Correspondence to: R.A. Latour; e-mail: LatourR@clemson.edu

Contract grant sponsor: NSF; contract grant number: BES-9986549

© 2004 Wiley Periodicals, Inc.

ities investigated were CH3, OH, COO�, and NH3�.

Further studies were then conducted to measure theability of integrins, fibroblast cells, and fibronectin-specific monoclonal antibodies to bind to the adsorbedfibronectin in order assess how adsorption may haveinfluenced binding site availability.4 Their datashowed that the amount of fibronectin adsorbed to theSAM surfaces followed the trend NH3

� � CH3 �COO� � OH, thus indicating that the positivelycharged NH3

� surface interacted most strongly withFn and the neutral hydrophilic OH surface the least.The monoclonal antibody binding results correlatedwell with integrin binding and cell adhesion func-tional assays, with binding affinity and cell adhesionstrength to the adsorbed fibronectin on the SAM sur-faces ranking according to the trend OH � COO� �NH3

� � CH3. These results thus indicated that Fnadsorbed onto the OH-SAM surface had the highestactivity, therefore suggesting the lowest degree of con-formational change, while the CH3-SAM surface in-duced the greatest degree of conformational change.These studies clearly demonstrate that the orientationand/or conformation of adsorbed fibronectin is pro-foundly influenced by surface chemistry, and raiseintriguing questions regarding how the molecularstructure of adsorbed fibronectin is actually altered bythe adsorption process. These questions, however, arevery difficult to answer using available experimentaltechniques. Additional methods are therefore neededto study protein adsorption behavior at the molecularlevel such that detailed analysis of adsorption inducedchanges in protein structure is possible.

Molecular modeling is perhaps the best currentlyavailable method to directly study molecular-level in-teractions. Advances in computational power and mo-lecular simulation techniques have led to the develop-ment of a wide variety of force field-based molecularmodeling programs that can potentially yield accuratequantitative information for the study of protein ad-sorption.7–12 Several studies investigating protein andpeptide adsorption to synthetic surfaces have beenconducted over the past decade using a wide varietyof approaches. Lu13 and Noinville et al.14 have con-ducted molecular mechanics simulations using vari-ous implicit solvation approximations to investigatethe effects of adsorption on protein conformation andorientation, respectively, while Nordgren et al.15 haveconducted molecular dynamics simulations of proteinadsorption on a surface that included 500 explicitlyrepresented water molecules to represent a condensedhydration layer. As is apparent in each of these stud-ies, the treatment of solvation effects is one of thebiggest challenges in the simulation of protein adsorp-tion behavior. The difficulties in the direct treatmentof solvation effects is clearly illustrated in a study byBujnowski and Pitt,16 in which the adsorption behav-ior of a simple 600-Da pentapeptide, enkephalin, on a

polyethylene surface was simulated using repeatboundary conditions and explicitly represented watermolecules. Even for this very simple system, about1500 water molecules (4500 atoms) had to be includedto adequately represent the solvent. Accordingly, pre-liminary studies showed that to fully solvate a systemincluding the full FnIII7–10 segment would result in asystem with over 100,000 atoms. The extremely largesize of such a simulation can be prohibitively expen-sive computationally, even for today’s powerful com-puter systems. Alternative approaches are thusneeded to enable the adsorption behavior of proteinsin an aqueous environment to be efficiently simulated.

The objectives of our research were therefore todevelop a computationally efficient molecular me-chanics-based approach that implicitly includes bulksolvation effects that can be applied to simulate theadsorption behavior of a protein on a functionalizedsurface, and then to demonstrate the application of thedeveloped force field in short molecular dynamicssimulations to theoretically investigate how surfacechemistry influences the initial adsorption behavior ofFnIII7–10 for comparison with the experimental resultsof Garcia et al.4,6 Force field development and modi-fication for nonbonded interactions (e.g., van derWaals and electrostatic interactions) is typically ac-complished by adjusting appropriate force field pa-rameters for pairwise atom-to-atom interactions suchto cause the molecular system of interest to reproduceexperimentally determined properties or the results ofquantum mechanical calculations. Because experimen-tal data currently do not exist in the form needed forour purposes, our approach to this problem was tomodify an existing force field, CHARMm, based onpreviously conducted semiempirical quantum me-chanical (SEQM) studies that described the energyversus distance relationships between individual pep-tide residues and functionalized surfaces that implic-itly includes solvation effects.17–19 The modified forcefield was then used in both molecular mechanics anddynamics (MM/MD) simulations to predict the orienta-tion and conformation of FnIII7–10 as it adsorbed onsurfaces with the same functionalities as used experi-mentally by Garcia et al.,4,6 but without requiring watermolecules to be explicitly represented in the simulation.The simulation results predict that the FnIII7–10 segmentadsorbs to each different surface type in a distinctlydifferent manner, with the results showing excellentagreement with the experimentally observed trends.

MATERIALS AND METHODS

Computational environment

All calculations were performed on an SGI O2 worksta-tion, networked to a more powerful SGI Dual Rack Onyx2

EFFECT OF SURFACE CHEMISTRY ON ADSORPTION OF A FIBRONECTIN FRAGMENT 687

Infinite Reality System, using the InsightII molecular mod-eling software (Accelrys, San Diego CA). InsightII is a mod-ular software package that incorporates several differentMM/MD force fields and analysis programs. For our calcu-lations we utilized version 27 of the CHARMm program andforce field. CHARMm was chosen for its recognized effi-ciency and accuracy in modeling intraprotein interactions,as well as its built-in features that allow easy modification ofthe force field equation. Analysis of the resulting structureswas performed using the Decipher and Analysis moduleswithin InsightII.

Materials

Alkanethiol self-assembled monolayers (SAMs) on goldwere chosen for this study to match the surfaces used ex-perimentally by Garcia et al.4,6 These surfaces are ideal formodeling studies because they have been very well charac-terized structurally, thus providing the necessary informa-tion for the development of representative molecular mod-els. Surfaces for this study were modeled after SAMs withfour different terminal functionalities, methyl-terminated(CH3-SAM), hydroxyl-terminated (OH-SAM), amine-termi-nated (NH3

�-SAM), and carboxyl-terminated (COO�-SAM).

In the experimental studies by Keselowsky et al.4 that wesought to represent in this molecular modeling study, theadsorption behavior of whole fibronectin was investigated.However, Fn is a very large protein (480 kDa); thus, it wouldbe very difficult to attempt to model the protein in itsentirety using current-day computational capabilities. Sub-sequent studies by Michael et al.6 have shown that theadsorption behavior and bioactivity of FnIII7–10 are verysimilar to that of whole Fn. Therefore, we chose to modelonly the 7–10 type III repeats of fibronectin (39-kDa seg-ment), which contain the putatative binding sites for �5�1

integrin; RGD and PHSRN. The crystal structure of FnIII7–10

was obtained from the Protein Data Bank (PBDID: 1fnf). Theninth repeat contains the PHSRN sequence, known as thesynergy site, and the 10th repeat contains the RGD loop. Thespatial position and structure of these domains have been

shown experimentally to be involved in integrin binding tofibronectin,20,21 and thus adsorption induced effects in thesedomains can be expected to significantly affect integrinbinding. Figure 1 presents the structure of the FnIII7–10,which is shaded as a function of the electrostatic potential atthe surface of the protein. As shown in this illustration, apositively charged domain exists on the protein’s surfacethat extends through both the RGD and PHSRN sequencesof this protein segment with the rest of the surface generallyexhibiting a negative charge. This crystal structure is widelyaccepted as an accurate structure, and has been used inprevious molecular modeling studies on Fn.22,23

Molecular modeling approach

Because of the large number of atoms involved, the sim-ulation of the adsorption of a protein to a synthetic surfacerequires the use of an empirical force field-based modelingapproach as opposed to the use of ab initio-based methods,which are typically restricted to the treatment of no morethan a few hundred atoms. An empirical force field is amathematical expression describing the dependence of thepotential energy of a molecule on the coordinates of theatoms in a system relative to a defined zero energy state.10,11

The force field equation is comprised of different mathemat-ical terms that define the physical interactions between at-oms in a system. The total potential energy calculated bymost force fields (Etotal) is the sum of all of the energy termsas indicated by Equation (1):

Etotal � Ebonds � Ebend � Etorsion � Eimp � EvdW � Eelec (1)

where the terms of the equation express the total potentialenergy of the system as the sum of individual potentialenergy contributions from bond stretching, angle bending,torsion bending, improper dihedral, van der Waals, andelectrostatic interactions, respectively.11 Each term in theequation is a computationally efficient mathematical expres-sion based on a set of user-defined force field parameters.The values of these parameters are established by first ac-quiring accurate data regarding individual pieces of the

Figure 1. Electrostatic potential map of FnIII7–10 at 310 K. Dark Gray (blue) � 0.7 kT/e, Light gray (red) � �-0.7 kT/e; k isBoltzman’s constant, T is absolute temperature (K), and e is the absolute value of the unit charge of an electron. [Color figurecan be viewed in the online issue, which is available at www.interscience.wiley.com.]

688 WILSON ET AL.

system being studied; particularly data from experimentalstudies and quantum mechanical calculations. The parame-ters of the force field are then adjusted until simulationsreplicate that data as accurately as possible.

As described in the introduction, the explicit representa-tion of water molecules in a molecular dynamics simulationof protein adsorption can be prohibitively expensive com-putationally, even for force field-based methods. As an al-ternative, implicit solvation methods, or continuum models,are often employed to represent the influence of bulk solventon the behavior of an explicitly represented solute–surfacesystem. This can be accomplished by incorporating the en-ergetic contributions of solvation directly into the force fieldpotential equation for the molecular system being simulated.

Based on the conditions of our particular system, wefound it necessary to develop a new method to implicitlyinclude solvation effects for our protein adsorption simula-tions. We based our approach on SEQM peptide–surfaceadsorption results obtained from previously conductedstudies by Latour et al.17–19 These previous studies com-bined both electrostatic and nonelectrostatic (i.e., hydropho-bic) solvation effects in the calculation of energy versussurface-separation-distance (SSD) profiles for selected pep-tide residue–SAM surface systems. To implement these re-lationships in our MM and MD simulations, we defined newatom types in the CHARMm force field to represent func-tional groups for each of our four SAM surfaces, and thenonbonded force field parameters of each of these new atomtypes were then adjusted until they were able to providepeptide–surface energy versus SSD behavior that closelymatched the SEQM simulation results.

Surface force field

Surfaces for this study were derived from a model of amethyl-terminated SAM surface [CH3(CH2)7CH3] [Fig. 2(a)]employed by Latour et al. in previous work investigatingmidchain residue adsorption,17–19 which was derived from amethyl-terminated alkanethiol SAM surface [S(CH2)15OCH3] on a [111] gold surface. The coordinates for our modelsurfaces were generated by truncating the SAM to retainonly the terminal methyl group of the SAM surface and

patching the broken COC bonds with hydrogens to satisfyvalence requirements [Fig. 2(b)]. The surfaces were theniteratively replicated until sufficient surface area was at-tained to support the fibronectin fragment model. Fixedconstraints were then applied to surface residues to preventthem from changing position. Following these methods, fouridentical surface planes were created to serve as templatesfor the later superposition of the appropriate surface forcefield potentials that would then endow each surface with theadsorption characteristics of one of four functionalities(CH3, OH, NH3

�, or COO�). Although it might appear thatthe truncation of the SAM surface in this way as unrealistic,truncation of the SAM surface to represent the interaction ofonly the top few atoms of the surface with the proteinactually results in a relatively small amount of error becausethe truncated nonpolar CH2 groups below the surface willexhibit primarily only van der Waals (vdW) forces, whichare typically considered negligible beyond a distance of 10Å. Furthermore, the effects of surface truncation is furtherminimized because there should be very little differencebetween the vdW forces exhibited between the truncatedCH2 groups and the atoms of the protein compared to atomsof water (which is implicitly represented). Very little wouldchange in terms of electrostatic interactions also, as only thetermini of the SAM chains contain charged species. Becauseany association between the idealized surfaces of this studyand a protein would only involve nonbonded interactionsbetween the functionalized groups of the surface and theatoms making up the protein, the only force field parametersthat needed to be modified to change the adsorptive char-acteristics of each surface were those related to the van derWaals and electrostatic terms.

For our calculations the surface atoms were treated essen-tially as united atom groups. Although the hydrogens of theterminal methyl groups were explicitly modeled in the co-ordinate files and exhibited repulsive forces correspondingto Pauli’s exclusion principle, all other force field contribu-tions were evaluated via the central carbon atom. Accord-ingly, the charge state of the atoms of the surface groupswere set to represent the surface charge of the correspondingsurface functional group that it served to represent (CH3 �0 e, OH � 0 e, NH3

� � 1 e, COO� � �1 e, where �1 erepresents the unit charge of an electron).

Figure 2. Image of methyl SAM surface used to create surfaces for force field parameterization: (a) original methyl SAMsurface, (b) truncated methyl SAM surface, (c) repeat unit of SAM surface with spacing of 5.0 Å. [Color figure can be viewedin the online issue, which is available at www.interscience.wiley.com.]

EFFECT OF SURFACE CHEMISTRY ON ADSORPTION OF A FIBRONECTIN FRAGMENT 689

The force field representing the energy contributions fromsurface–protein interactions was a combination of two sim-ple, computationally efficient, equations. The electrostaticinteraction of the surface with the peptide residues of theprotein was represented by a standard Coulomb potential,as presented in Equation (2):

Eelec � �i�1

N�1 �j�i�1

N qiqj

4�orij(2)

where the interaction energy between two atoms is a resultof the product of the two atomic charges (qi and qj) dividedby the separation distance between them, rij, in a mediumwith a permittivity of free space, o, and a relative dielectricconstant, . This equation is used by most force fields torepresent electrostatic interactions.17 In this study, all calcu-lations were performed with a continuum dielectric equal tothat of water ( � 78.9) as a simple method of implicitlyrepresenting an aqueous environment that screens interac-tions between charged residues. The second equation, theLennard-Jones (6, 12) potential, was chosen to model thecombined contributions due to both van der Waals interac-tions and hydration effects [Equation (3)]

ELJ � �i�1

N�1 �j�i�1

N

r�� rm

rij� 12

� 2� rm

rij� 6� (3)

where rij is the distance between atoms i and j, r is theminimum energy of the potential curve, and rm is the sepa-ration distance for which the energy is the minimum of thepotential curve. This expression for the van der Waals inter-actions was chosen for two reasons. First, SEQM data incor-porating implicit solvation show that the adsorption profilesof peptide residues on SAM surfaces display an SSD rela-tionship that can be effectively represented with a Lennard-Jones (6–12) potential. Second, preexisting commandswithin InsightII allow for easy application of this functionand manipulation of the rm and r, parameters to adjust theforce field potential of each surface type to closely representthe previously determined SEQM data.

The parameters rm and r were determined for pairwiseinteractions by an iterative process of trial and error (TableI). To accomplish this, selected systems of individual peptideresidues and surfaces similar to those in work by Latour etal.17–19 were constructed. The residues selected for the pa-rameterization of the force field were alanine, arginine, andaspartic acid, which represented hydrophobic, positivelycharged, and negatively charged residues, respectively.Each residue was modeled over each of the four types ofSAM surfaces to replicate the systems used by Latour and

coworkers.17–19 The enthalpies of the systems were calcu-lated as a function of SSD. SSD was evaluated as done byLatour et al., where residues/surface systems were designedto represent peptide residues protruding from a proteinsurface and SSD was measured from the terminal atom ofthe residue functional group to the plane of the surface asdefined by the plane of the central carbon atoms of thesurface groups. The parameters rm and r were then adjustedfor each atom type to recreate an energy profile as close tothat of the previously determined SEQM data as possible.Figure 3 shows examples of each characteristic residue–surface system: hydrophobic, hydrophilic, like charge, andopposite charge, respectively. From these comparisons, it isshown that the modified CHARMm force field is able toclosely represent the SEQM results for the hydrophobic,hydrophilic, and oppositely charged systems, with the re-pulsion effects for the same-charged system being somewhatunderestimated. Although currently there are no experi-mental data that can be used for the direct verification of thecalculated values, the energy versus SSD plots described bythe previous SEQM studies, and the subsequent modifiedCHARMm force field parameters, express relationships foreach type of molecular interaction that we believe are intu-itively very reasonable.

Selection of initial protein orientations on surfaces

Once the force field had been modified, it was necessary todetermine orientations of the FnIII9–10 segment that pro-vided the most energetically favorable interaction, to deter-mine which orientations were to be further studied usingmolecular dynamics simulations. We used only the 9–10subunits for these calculations, as we were most interestedin how adsorption would affect the integrin binding do-mains contained in these subunits. To do this, we prepareda model of the FnIII9–10 segment repeats from the largerFnIII7–10 fragment by truncating the glycine linker betweenthe eighth and ninth subunits. As shown in Figure 4, theFnIII9–10 fragment was then oriented above the surface suchthat the long axis of the protein was parallel with the planeof the SAM surface. The initial SSD was set to be 18 Å fromthe center of mass of the protein segment to the plane of thesurface. This initial SSD is somewhat arbitrary, and wasdetermined by trial and error. It was important to set theinitial surface separation distance such that the proteincould be rotated about its major axis and not pass through orcollide with the SAM surface. Direct contact between theprotein and the model surface would yield artifactually highenergies that would not reflect a realistic state.

TABLE ILennard-Jones Parameters Used in Modified Force Field

CH3-SAM OH-SAM NH3�-SAM COO�-SAM

Cεr �3.6 kcal/mol �0.001 kcal/mol �0.75 kcal/mol �0.18 kcal/molrm 2.5 Å 4.6 Å 4.0 Å 4.0 Å

Nεr �0.001 kcal/mol �0.001 kcal/mol �0.46 kcal/mol �0.051 kcal/molrm 5.5 Å 5.0 Å 5.4 Å 8.0 Å

Oεr �0.001 kcal/mol �0.001 kcal/mol �0.01 kcal/mol �0.001 kcal/molrm 9.5 Å 7.0 Å 8.4 Å 8.0 Å

690 WILSON ET AL.

Figure 3. Plots of energy versus surface separation distance for selected residue–surface systems. SEQM results on right,modified CHARMm force field on left: (a) alanine with a CH3-SAM surface, (b) arginine with an OH-SAM surface, (c) argininewith a COO�-SAM surface, (d) aspartic acid with a COO�-SAM surface.

EFFECT OF SURFACE CHEMISTRY ON ADSORPTION OF A FIBRONECTIN FRAGMENT 691

The �-carbon of Asp354, the apex of the RGD loop, waschosen as a reference point for rotation of the protein aboutits long axis. The degree of rotation about the protein axiswas defined by the vector extending perpendicularly fromthe major axis of the protein segment and passing throughthe �-carbon of Asp354. Zero degrees rotation was defined asbeing the orientation when this vector was normal to thesurface plane (Fig. 4). The protein segment was then rotatedin 15° increments about its long axis (i.e., axis parallel to thesurface plane) over a full 360°, and the system energy wascalculated using a molecular mechanics energy minimiza-tion calculation at each position to sample the energy spacefor the initial protein/surface interaction. For each of theinitial configurations generated, the system was minimizedusing a combination of the steepest descents and adopted-basis Newton-Raphson algorithm, 10,000 steps each, to cal-culate the energy of the entire system. The spherical cutoffradius was set to 20 Å. The resulting energies were plottedas a function of rotation about the protein axis (Fig 5), andwere then used to predict the low-energy orientation of theFnIII9–10 segment on the surface. These energy values rep-

resent the difference in system energy when the protein islocated over the surface at a designated orientation relativeto the energy of the protein and surface when they areinfinitely separated. These energy effects are due to contri-butions from both intermolecular interactions (e.g., van derWaals and electrostatic interactions) and intramolecular in-

Figure 4. Ninth and 10th type III repeats of fibronectin ona model surface with RGD and PHSRN sites labeled andshaded in lighter color (top). Illustration of frame of refer-ence use for rotation of the 9th and 10th type III repeats of Fnon model surfaces (bottom). [Color figure can be viewed inthe online issue, which is available at www.interscience.wiley.com.]

Figure 5. Energy versus rotation angle for Fn9–10 on (a)model OH-SAM surface, (b) model CH3-SAM surface, (c)model NH3

�-SAM surface, (d) on model COO�-SAM sur-face.

692 WILSON ET AL.

teractions (e.g., conformational changes within the mole-cules). A negative energy represents a condition in whichadsorption lowers the energy of the system, representing afavorable interaction, while a positive energy value indicatesan adsorbed orientation that is energetically unfavorablerelative to the nonadsorbed state. This procedure was re-peated for all four model surfaces.

Molecular dynamics and analysis

After low-energy orientations had been determined forFnIII9–10, the corresponding minimized structures wereused to perform short molecular dynamics calculations withthe full FnIII7–10 protein segment to generate theoreticalpredictions of the adsorbed conformation. Calculations wereperformed using a pair-list Verlet algorithm with a 1.0-fstime step, and a spherical cutoff radius of 20 Å. The systemswere heated to 310 K over 400 fs and allowed to equilibratefor a minimum of 50 ps. After molecular dynamics simula-tions were complete, the resulting structures were evaluatedto assess adsorption-induced changes in the protein seg-ment.

RESULTS AND DISCUSSION

Figure 5 shows the energy versus rotation angleplots for the molecular mechanics calculations. Eachsystem shows a distinct energy profile with multiplemaxima and minima. The CH3-SAM model surfaceshowed only two points with favorable adsorptionenergies (i.e., energy less than 0 kcal/mol), the deepestof which is located a 0° rotation about the axis of thefragment with an adsorption energy of �38 kcal/mol.The energy of the second orientation (75° rotation) isclose to zero, thus indicating a much weaker interac-tion. This result is interesting in that it suggests that onhydrophobic surfaces Fn is likely to adsorb in anorientation such that the integrin binding domains aremost accessible to membrane bound integrins. In allother orientations it is predicted that adsorption isinhibited by strongly hydrophilic residues on the pro-tein surface that preferentially are attracted to watermolecules rather than the functional groups of thesurface. In comparison, the OH-SAM exhibits a num-ber of adsorption orientations that are competitive,lying within a few kT of the global minimum at 0°,with the global minimum energy value being zerokcal/mol at 0° rotation The NH3

�-SAM surface showsits lowest minimum at 15° rotation. Thus, the mostfavorable adsorbed orientation is predicted to be onein which the integrin binding sites are accessible witha corresponding adsorption energy of �72 kcal/mol.This system is largely dominated by electrostatic in-teractions, which is to be expected, because theFnIII9–10 has a strongly positive electrostatic region

running through the integrin binding domains (asshown in Fig. 1), which causes a strong repulsiveenergy over the range of 105 to 255° that would the-oretically resist the adsorption of the FnIII9–10 frag-ment with this orientation to the surface [Fig. 5(c)].The COO�-SAM system is also dominated by electro-static interactions. In contrast to the NH3

�-SAM sur-face, our calculations show that FnIII9–10 will prefer-entially adsorb when oriented at 90 to 195° on thenegatively charged COO�-SAM surface with an ad-sorption energy of �37 kcal/mol. This suggests thatthe RGD and PHSRN sites would be less accessible tointegrin binding when Fn is adsorbed on this surface.

Using these initial data, we selected the minimumenergy orientation from each of the protein/surfacesystems for molecular dynamics to represent examplesystems with high probability of occurrence comparedto other adsorbed orientations. Figures 6–10 show theresulting structures of FnIII7–10 after 50 ps of molecu-lar dynamics simulations, with the initial conforma-tion of FnIII7–10 shown in gray and the final structureafter 50 ps in white. The first image, shown in Figure6, was produced using the unmodified CHARMmforce field for the CH3-SAM surface, and with � 78.9,as a baseline for comparison to the modified forcefield. As expected with the unmodified force field allcontacting regions of the protein were weakly at-tracted to the model surface and denatured slightly.This occurs due to the fact that under these simulationconditions all residue–surface interactions are attrac-tive until interacting atoms approach within their vander Waals radius and there are no competing hydra-tion effects. Results from the modified force field forthis same system were quite different, however, asshown in Figure 7. In contrast to the behavior on theunmodified CHARMm surface, FnIII7–10 adsorbedtightly with the model CH3-SAM surface, but only inthe 10th domain, thus suggesting the presence of ahydrophobic patch on the Fn surface at this location. Itis evident in Figure 7 that the 10th type III subunit ofFn closely associates with the model surface while theseventh to ninth segments maintain a surface separa-tion distance that is indicative of a retained hydrationlayer, thus making these segments resistant to surfaceadsorption. It can be seen that a large degree of dena-turation has taken place in the 10th subunit for theinternal residues to come into intimate contact withthe surface. This is very similar to what is believed tooccur when proteins adsorb onto strongly hydropho-bic surfaces.24–27 Due to water’s strong tendency toassociate by hydrogen bonding, it is energetically fa-vorable for hydrophobic species to adsorb together inan aqueous environment to reduce their solvent-acces-sible surface area (SASA). By reducing its SASA, aprotein can maintain a more stable structure in aque-ous solution. When a protein comes into contact witha hydrophobic surface, however, these hydrophobic

EFFECT OF SURFACE CHEMISTRY ON ADSORPTION OF A FIBRONECTIN FRAGMENT 693

residues can separate from each other and adsorb tothe hydrophobic surface, thereby further reducing theSASA of the entire system.10 It is anticipated that

longer simulations will show more dramatic changesin the protein’s structure on the CH3-SAM surfacecompared to the other surfaces, because of its greater

Figure 6. Image of initial conformation (dark gray, blue) and resulting conformation of FnIII7–10 after 50 ps of moleculardynamics on model CH3-SAM surface (white) using the unmodified CHARMm force field. Asterisks (*) indicate location ofRGD, pound sign (#) indicates location of PHSRN. [Color figure can be viewed in the online issue, which is available atwww.interscience.wiley.com.]

Figure 7. Illustration of initial conformation (dark gray, blue) and resulting conformation of FnIII7–10 after 50 ps of moleculardynamics on CH3-SAM surface (white) using modified CHARMm force field. Asterisks (*) indicate location of RGD, poundsigns (#) indicate location of PHSRN. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

694 WILSON ET AL.

potential to strongly interact with the protein’s innerhydrophobic core. A simulation time of 50 ps is notnearly long enough to allow the protein to open up to

fully expose its hydrophobic core residues to the sur-face, and achieve the maximum denaturation possible.The primary objectives of this present study, however,

Figure 8. Illustration of initial conformation (dark gray, blue) and resulting conformation of FnIII7–10 after 50 ps of moleculardynamics on OH-SAM surface (white) using modified CHARMm force field. Asterisks (*) indicate the location of RGD, thepound sign (#) indicates location of PHSRN. [Color figure can be viewed in the online issue, which is available atwww.interscience.wiley.com.]

Figure 9. Illustration of initial conformation (dark gray, blue) and resulting conformation of FnIII7–10 after 50 ps of moleculardynamics on NH3

�-SAM surface (white) using modified CHARMm force field. Asterisks (*) indicate location of RGD, poundsign (#) indicates location of PHSRN. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

EFFECT OF SURFACE CHEMISTRY ON ADSORPTION OF A FIBRONECTIN FRAGMENT 695

were to explore the concept of parameterizing an ex-isting force field to reproduce SEQM data that in-cluded implicit solvation effects, and to apply that to aprotein adsorption system to investigate its initial ad-sorption behavior. Longer molecular dynamics stud-ies must be conducted to more fully explore the abilityof the developed protein adsorption force field to sim-ulate structural changes in the protein following initialadsorption.

As seen in Figure 8, the FnIII7–10 fragment did notadsorb onto the model OH-SAM surface. When com-pared to the initial configuration it is evident that theentire fragment has translated away from the surface.This is relatively similar to what is found to occurexperimentally on OH-SAM surfaces, which can becharacterized as being neutral hydrophilic. Experi-mental studies, however, typically do show some de-gree of adsorption on OH-SAM surfaces, although itcan be questioned whether or not this is due to theunavoidable small percentage of defects in such asurface or due to the functionality itself. The lack ofany adsorption in the model results thus represents anextreme case for the OH-SAM surface. The predictedadsorption behavior is a direct consequence of theforce field parameterization. Refinement may beneeded here to more appropriately simulate a smalldegree of intramolecular attraction in the OH-SAMsurfaces, perhaps due to the formation of hydrogenbonds between peptide residues and the surface that

are more stable than either the residues or surfacegroups can form with water.

Figures 9 and 10 show the resulting Fn conforma-tions for adsorption onto charged SAM surfaces(NH3

� and COO� terminated SAMs). Evaluation ofpredicted FnIII7–10 adsorption behavior on the NH3

�-SAM surface shows that each of the 7–10 segments ispredicted to be strongly adsorbed with a moderatedegree of subsequent denaturation. In contrast to this,only the 9–10 segments are adsorbed on the COO�-SAM surface, but with little apparent structural dis-turbance. In both of these systems it is interesting tonote that although the proteins are clearly adsorbed tothe surface, they do not come into close contact withthe surface; rather, they maintain a hydration layerbetween themselves and the surface. This effect, whichresults from the SEQM binding curves to which themodified potential was fit, is indicative of the “elec-trostatic trapping” effect that has also been observedexperimentally. Work by Xu et al.,28 in which themotion of single dye-labeled proteins was trackedover time, demonstrated that long-range electrostatictrapping is the dominant driving force for proteinadhesion on charged surfaces.

In summary, the initial adsorption energy resultspresented in Figure 5 predict that the amount of Fnadsorbed to the four surfaces should be in the order ofNH3

� � CH3 � COO� �� OH, with the adsorbedorientation being such that the integrin binding sites

Figure 10. Illustration of initial conformation (dark gray, blue) and resulting conformation of FnIII7–10 after 50 ps ofmolecular dynamics on COO�-SAM surface (white) using modified CHARMm force field. Asterisks (*) indicate location ofRGD, pound sign (#) indicate location of PHSRN. [Color figure can be viewed in the online issue, which is available atwww.interscience.wiley.com.]

696 WILSON ET AL.

are readily accessible for each of the surfaces exceptfor COO�. From the molecular dynamics simulationsand analyses, the amount of adsorption-induced de-naturation is predicted to rank in the order of NH3

� �CH3 � COO� � OH. We believe, however, that withsufficient simulation time the CH3-SAM surfacewould ultimately show a higher degree of denatur-ation than the NH3

�-SAM. These overall results sug-gest that the charged surfaces should each exhibitsome degree of decreased integrin binding capabilityfollowing adsorption but for different reasons; theNH3� surface due to adsorption induced structuraldistortion and the COO� surface due to unfavorableadsorbed orientation. Because of the minimal interac-tions of the FnIII7–10 fragment with the OH surface, anyadsorbed FnIII7–10 should exhibit very minimal degreesof denaturing, thus retaining its bioactivity for integrinbinding. In contrast, the CH3-SAM surface should beexpected to be the most denatured (given sufficient sim-ulation time), with the greatest loss of its integrin bind-ing capability. These trends show excellent agreementwith the SPR and Enzyme-linked Immunosorbent Assay(ELISA) experimental results reported by Garcia et al.4,6

and provide plausible molecular-level explanations forthe observed experimental behavior.

At this point it is clear that our new force field does notcompletely encompass all elements involved in proteinadsorption. For example, one type of interaction that hasnot been accounted for, which is known to be important,is lateral protein/protein interactions. However, by re-ducing solvent degrees of freedom as we have, it shouldbe possible to study even larger surface/protein systemswith multiple protein fragments. The study of such mul-tiple protein/surface systems would simply not be pos-sible at this time if solvent degrees of freedom wereexplicitly included. Additionally, the length of the sim-ulation (50 ps) is not sufficient to completely model theadsorption process, but was used in this project to dem-onstrate the implementation of the modified CHARMmforce field in an MD simulation, with the development ofthis modified force field representing the primary focusof this work. The consideration of such short simulationtimes is still very useful, however, as it provides insightinto the initial events of protein adsorption and subse-quent trends for surface-induced changes in adsorbedconformation of this protein segment that may help usunderstand the experimentally observed behavior. Fu-ture work will focus on performing longer more rigor-ous simulations on multiple adsorption orientations (i.e.,all energy-favorable minima) to better represent actualadsorption events.

CONCLUSIONS

In this research a new surface force field with im-plicit solvation was developed for the study of protein

adsorption to synthetic surfaces. This force field wasparameterized to incorporate SEQM molecular mod-eling data regarding the adsorption behavior of indi-vidual midchain peptide residues on SAM surfaces inan aqueous environment and then applied to investi-gate the initial adsorption behavior of a fibronectinsegment containing integrin binding domains. The re-sults of these simulations show distinctly differentadsorption behaviors for FnIII7–10 on each surface thatare in qualitative agreement with experimental stud-ies. These simulations suggest molecular-level mech-anisms for the observed experimental behavior thatprovide insights into the possible molecular behaviorduring the initial stages of adsorption. The resultsfrom this study indicate that, while still in the earlystages of development, an important step forward hasbeen taken in developing a computationally efficientforce field that is specifically designed for studying theadsorption behavior of large proteins on syntheticsurfaces. By excluding solvent degrees of freedom itwas possible to reduce the size of the system by anorder of magnitude (from 100,000 atoms to 10,000atoms). This is very significant, as calculation time fornonbonded interactions, such as protein/surface in-teractions, is directly proportional to the number ofatoms in the system.18 In terms of real-life proteinadsorption studies, however, the systems consideredin our calculations are still very small, and simulationtimes are very short. Thus, faster and more efficientcomputational strategies are still required. Further de-velopment of these methods, combined with furtherexperimental studies for verification of results, shouldprovide the necessary tools to enable biomaterial sur-faces to be designed to proactively control the orien-tation and conformation of adsorbed proteins andthereby provide a means to directly control cellularresponse for improved biocompatibility.

The authors would like to thank Drs. Robert Funchess andKen Butenhoff of Accelrys technical support and Mr. GaryGrahek of the Department of Bioengineering, Clemson Uni-versity, for their help in troubleshooting problems that aroseduring the course of this study. Also, we would like thethank Corey Ferrier for her invaluable assistance as thesystems administrator of the VRNet and the Center forAdvanced Engineering Films and Fibers (CAEFF) at Clem-son University for computation resources support.

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