pubs.acs.org/Langmuir © 2010 American Chemical Society
Molecular Understanding of Conformational Dynamics of a Fibronectin Module on Rutile (110) Surface Chunya Wu, Mingjun Chen,* and Cheng Xing Center for Precision Engineering, Harbin Institute of Technology, P.O. Box 413, Harbin 150001, China Received July 28, 2010 The conformational dynamics of the 10th type-III module of fibronectin (FN-III10) adsorbed on the perfect and three reduced rutile TiO2(110) surfaces with different types of defects was investigated by molecular dynamics (MD) simulations. Stable protein-surface complexes were presented in the four simulated models and were derived from the contributions of direct and indirect interactions of various functional groups in FN-III10 with the metal oxide layers. A detailed analysis to characterize the overall structural stability of the adsorbed FN-III10 molecule suggests that the bonding strength and the loss of protein secondary structure vary widely, depending on the topology of the substrate surface. The additional adsorption sites exhibiting higher activity, provided by the reduced surfaces, are responsible for the stronger FN-III10-TiO2 interactions, but too high an interaction energy will cause a severe conformational deformation and therefore a significant loss of bioactivity of the adsorbed protein.
I. Introduction Interfacial interactions between proteins and biomaterials have an important effect on the cellular response in biological environments. Because protein will be adsorbed from body fluids before cells adhere to the surface of a biomaterial, this influences the subsequent cell behavior.1 When the biomaterial is suddenly placed in a biological milieu containing cells, integrin receptors located at the cell membrane will actively search for the specific ligands to bind with the surface. If the ligands on the surface maintain a defined density and certain conformations to expose the ligand-receptor binding contacts, then good cell-biomaterial response may be induced successfully.2 Hence, the key to the cell acceptance of biomaterial as the homologue is the existence of specific ligands on the surface, which can be identified by integrin, such as several extracellular matrix (ECM) proteins, among which fibronectin (FN), comprising three different kinds of homology units referred to as types I-III3 (Figure 1), is a typical one. The role of FN in cell adhesion, migration, and proliferation has already been extensively documented in the literature.4-6 Yang7 seeded osteoblasts onto Ti and FN preadsorbed Ti surfaces. He found the presence of FN to be an important factor *To whom correspondence should be addressed. Tel: þ86 (0)451-86403252. Fax: þ86 (0)451-86403252. E-mail:
[email protected].
(1) Kasemo, B. Biological surface science. Surf. Sci. 2002, 500, 656–677. (2) Yao, K. D. Biomaterials Related to Tissue Engineering; Chemical Industry Press: Beijing, 2003. (3) Ruoslahti, E. Fibronectin and its receptors. Annu. Rev. Biochem. 1988, 57, 375–413. (4) Geiger, B.; Bershadsky, A.; Pankov, R.; Yamada, K. M. Transmembrane crosstalk between the extracellular matrix and the cytoskeleton. Nat. Rev. Mol. Cell. Biol. 2001, 2, 793–805. (5) Sogo, Y.; Ito, A.; Matsuno, T.; Oyane, A.; Tamazawa, G.; Satoh, T.; Yamazaki, A.; Uchimura, E.; Ohno, T. Fibronectin-calcium phosphate composite layer on hydroxyapatite to enhance adhesion, cell spread and osteogenic differentiation of human mesenchymal stem cells in vitro. Biomed. Mater. 2007, 2, 116–123. (6) Middleton, C. A.; Pendegrass, C. J.; Gordon, D.; Jacob, J.; Blunn, G. W. Fibronectin silanized titanium alloy: a bioinductive and durable coating to enhance fibroblast attachment in vitro. J. Biomed. Mater. Res., Part A 2007, 83, 1032–1038. (7) Yang, Y. Z.; Glover, R.; Ong, J. L. Fibronectin adsorption on titanium surfaces and its effect on osteoblast precursor cell attachment. Colloids Surf., B 2003, 30, 291–297.
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in controlling the interaction between the surfaces and living cells. Wittmer8 investigated the cell-attachment properties of FNterminated multilayer films composed of linear polyelectrolytes poly(L-lysine) and dextran sulfate. The experimental result indicated that cells spread to a greater extent and more symmetrically on films coated with a layer of FN. To provide reliable data for protein adsorption on solid surfaces, experimental approaches, such as surface tensiometry,9 ellipsometry,10 infrared spectroscopy,11 immunoassay,12 quartz crystal microbalance with dissipation (QCMD),13,14 and atomic roughnessforce microscopy (AFM),14-17 have been widely adopted in recent years. Sousa15 analyzed the dynamics of FN adsorption on two different titanium oxides, with varied surface roughness, assessed by radiolabeling, XPS, ellipsometry, and AFM. Hovgaard13 investigated the concentration effects and conformational changes in FN adsorbed on flat and nanometerscale-roughness tantalum oxide surfaces using ellipsometry and (8) Wittmer, C. R.; Phelps, J. A.; Saltzman, W. M.; Van Tassel, P. R. Fibronectin terminated multilayer films: protein adsorption and cell attachment studies. Biomaterials 2007, 28, 851–860. (9) Wang, J.; McGuire, J. Surface tension kinetics of the wild type and four synthetic stability mutants of T4 phage lysozyme at the air-water interface. J. Colloid Interface Sci. 1997, 185, 317–323. (10) Joshi, O.; Lee, H. J.; McGuire, J.; Finneran, P.; Bird, K. E. Protein concentration and adsorption time effects on fibrinogen adsorption at heparinized silica interfaces. Colloids Surf., B 2006, 50, 26–35. (11) Flach, C. R.; Brauner, J. W.; Taylor, J. W.; Baldwin, R. C. External reflection FTIR of peptide monolayer films in situ at the air/water interface: experimental design, spectra-structure correlations, and effects of hydrogendeuterium exchange. Biophys. J. 1994, 67, 402–412. (12) Yu, L.; Li, C. M.; Zhou, Q.; Luong, J. H. T. Poly (vinyl alcohol) functionalized poly (dimethylsiloxane) solid surface for immunoassay. Bioconjugate Chem. 2007, 18, 281–284. (13) Hovgaard, M. B.; Rechendorff, K.; Chevallier, J.; Foss, M.; Besenbacher, F. Fibronectin adsorption on tantalum: the influence of nanoroughness. J. Phys. Chem. B 2008, 112, 8241–8249. (14) Dolatshahi-Pirouz, A.; Jensen, T.; Foss, M.; Chevallier, J.; Besenbacher, F. Enhanced surface activation of fibronectin upon adsorption on hydroxyapatite. Langmuir 2009, 25, 2971–2978. (15) Sousa, S. R. Dynamics of fibronectin adsorption on TiO2 surfaces. Langmuir 2007, 23, 7046–7054. (16) Yu, L.; Lu, Z. S.; Gan, Y.; Liu, Y. S.; Li, C. M. AFM study of adsorption of protein A on a poly (dimethylsiloxane) surface. Nanotechnology 2009, 20, 1–6. (17) Hernandez, J. C. R.; Rico, P.; Moratal, D.; Pradas, M. M. Fibrinogen patterns and activity on substrates with tailored hydroxy density. Macromol. Biosci. 2009, 9, 766–775.
Published on Web 09/21/2010
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Figure 1. Schematic structure of the fibronectin monomer. Type I-III modules are represented by ellipses, triangles, and squares, respectively. The solution NMR configuration of FN-III10 is displayed in the bottom-right corner by a new cartoon mode combined with a line mode generated by VMD. The secondary structure of FN-III10 consists of two antiparallel β sheets containing seven β strands, labeled A-G. The RGD sequence lying close to the C terminus is shown in VDW mode.
QCMD. Dolatshahi-Pirouz14 examined the adsorption characteristics of fibronectin on hydroxyapatite and a reference gold substrate by means of QCMD and AFM. Progress has also been made with respect to the theoretical calculations of the thermodynamic and kinetic properties of the adsorbed proteins, especially in molecular dynamics (MD) simulation, which is capable of providing abundant molecular-level information. Tobias18 and Paggi19 performed MD simulations to study the behavior of cytochrome c on surfaces under different conditions. Monti20,21 inspected the early reaction processes taking place in the protein-mineral interface regions through the MD approach. Raffaini22 and Shen23 reported MD results of the adsorption dynamics of FN modules on graphite and hydroxyapatite surfaces, respectively. However, the adsorbents used were typically restricted to perfect surfaces without any defects, and little attention has been paid to the surface structure of the substrate, which should be of particular importance because the characteristics of the binding surface may modify the biological activity of protein related to subsequent cell adhesion.24,25 As is well known, the cell adhesion activity of FN has been localized to the Arg-Gly-Asp sequence (RGD, a tripeptide with a high specificity for integrin receptors) lying close to the C terminus of the 10th FN-III module (FN-III10).26,27 In this article, (18) Tobias, D. J.; Mar, W.; Blasie, J. K.; Klein, M. L. Molecular dynamics simulations of a protein on hydrophobic and hydrophilic surfaces. Biophys. J. 1996, 71, 2933–2941. (19) Paggi, D. A.; Martı´ n, D. F.; Kranich, A.; Hildebrandt, P. Computer simulation and SERR detection of cytochrome c dynamics at SAM-coated electrodes. Electrochim. Acta 2009, 54, 4963–4970. (20) Monti, S. Molecular dynamics simulations of collagen-like peptide adsorption on titanium-based material surfaces. J. Phys. Chem. C 2007, 111, 6086–6094. (21) Monti, S. RAD16II β-sheet filaments onto titanium dioxide: dynamics and adsorption properties. J. Phys. Chem. C 2007, 111, 16962–16973. (22) Raffaini, G.; Ganazzoli, F. Molecular dynamics simulation of the adsorption of a fibronectin module on a graphite surface. Langmuir 2004, 20, 3371–3378. (23) Shen, J. W.; Wu, T.; Wang, Q.; Pan, H. H. Molecular simulation of protein adsorption and desorption on hydroxyapatite surfaces. Biomaterials 2008, 29, 513–532. (24) Hederos, M.; Konradsson, P.; Liedberg, B. Synthesis and self-assembly of galactose-terminated alkanethiols and their ability to resist proteins. Langmuir 2005, 21, 2971–2980. (25) Liu, L. Y.; Chen, S. F.; Giachelli, C. M.; Ratner, B. D. Controlling osteopontin orientation on surfaces to modulate endothelial cell adhesion. J. Biomed. Mater. Res., Part A 2005, 74A, 23–31. (26) Alison, L. M.; Timothy, S. H. The three-dimensional structure of the tenth type III module of fibronectin: an insight into RGD-mediated interactions. Cell 1992, 71, 671–678. (27) Pierschbacher, M. D.; Ruoslahti, E. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 1984, 309, 30–33.
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we present a series of MD simulations on the conformational dynamics of the FN-III10 molecule adsorbed on rutile TiO2(110) surfaces in vacuum. To enrich the simulated substrate with the nature of the machined surfaces in the near future, special attention has been paid to the effect of surface defects on the adsorption behavior and conformational changes of the model protein.
II. Simulation Details Model Building. The high solution crystal structure of FNIII10 was derived from the Protein Data Bank (pdb id 1fna), made available to us by Dickinson.28 The structure of FN-III10 shown in Figure 1 contains seven β strands that form a sandwich of two antiparallel β sheets, one containing three strands (ABE) and the other containing four strands (CDFG). The triple-stranded β sheet consists of residues Glu9-Thr14 (A), Leu18-Asp23 (B), and Thr56-Ser60 (E), and the four-stranded β sheet comprises residues Tyr31-Glu38 (C), Gln46-Pro51 (D), Val66-Thr76 (F), and Ile88-Thr94 (G). The configuration of FN-III10 is displayed by the new cartoon mode combined with the line mode by VMD software, with the Arg78-Gly79-Asp80 sequence at the apex of the F-G loop highlighted by the VDW mode.29 Titanium, a biocompatible metal commonly used in medical implants, is known to have a naturally stable oxide layer on the surface, whose major constituent is titanium dioxide.30,31 Rutile is generally considered to be the most stable phase of TiO2 polymorphs under ambient conditions.32 As shown in Figure 2a, the rutile (110) perfect surface is characterized by rows of 5-fold Ti sites (Ti5) coordinated to the exposed 3-fold O atoms (O3) as well as rows of doubly coordinated bridging O atoms (Ob) protruding above the Ti-O plane and bound to two 6-fold Ti atoms (Ti6). To assess the influence of surface defects on the adsorption behavior of protein, we created three types of surface defects (28) Dickinson, C. D.; Veerapandian, B.; Dai, X. P.; Hamlin, R. C.; Xuong, N. Crystal structure of the tenth type III cell adhesion module of human fibronectin. J. Mol. Biol. 1994, 236, 1079–1092. (29) Humphrey, W.; Dalke, A. VMD-visual molecular dynamics. J. Mol. Graphics 1996, 14, 33–38. (30) Jones, F. H. Teeth and bones: applications of surface science to dental materials and related biomaterials. Surf. Sci. Rep. 2001, 42, 79–205. (31) Massaro, C.; Rotolo, P.; Riccardis, F. D.; Milella, E.; Napoli, A. Comparative investigation of the surface properties of commercial titanium dental implants. Part I: chemical composition. J. Mater. Sci.: Mater. Med. 2002, 13, 535– 549. (32) Dachille, F.; Simons, P. Y.; Roy, R. Pressure-temperature studies of anatase, brookite, rutile, and TiO2-II. Am. Mineral. 1968, 53, 1929–1939.
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Figure 2. Structure of rutile TiO2(110) surfaces. (a) Perfect surface, (b) partial view of the surface with point defects, (c) sectional view of the surface with a step defect, (d) sectional view of the surface with a groove defect. Table 1. Parameters of the Simulated Models parameters defect dimensions (A˚3) TiO2 FN-III10
model I
9500
model II
model III
O vacancy step 144.94 116.95 15.52 9500 9367 1
model IV groove 9300
(i.e., point, step, and groove) as well as a perfect rutile surface, followed by a series of MD simulations on the molecular models comprising one of the four rutile TiO2(110) surfaces and a FN-III10 module with an initial distance of >5 A˚ between the lowermost point of the model protein and the top Ti-O plane, that is, model I-IV (Table 1). The perfect surface placed in the x-y plane was created by the periodic replication of an elementary cell along the x and y directions, respectively, leading to a xy size of 144.94 116.95 A˚2. The point defects, that is, oxygen vacancies, were created by artificially shifting six Ob atoms to undercoordinated Ti sites from their initial positions, and the step and groove defects were produced by removing the top TiO2 layers of the perfect surface to form a rectangle (35.50 29.96 3.25 A˚2) and a V-shape slot (with a depth of 6.50 A˚), respectively. Figure 2b gives the partial view of a TiO2 surface with point defects, and the sectional views of the step and groove are presented in Figure 2c,d, respectively. Molecular Dynamics Simulation Protocol. A series of MD simulations using identical protocols were performed to simulate the adsorption behaviors of FN-III10 molecules on various rutile TiO2(110) surfaces at T = 310.15 K in the canonical ensemble (NVT) with a time step of 1 fs. TiO2 was reproduced through the Matsui and Akaogi parametrization,33 and the structure of FN-III10 was described by the AMBER force field34 using the SHAKE algorithm35 to constrain bonds connected to H atoms. Periodic boundary conditions were applied in the x and y directions, with a periodic vacuum gap of a 3-fold slab model thickness along the z direction. A cutoff of the nonbonded van der Waals force with a switching function starting at a distance of (33) Matsui, M.; Akaogi, M. Molecular dynamics simulation of the structural and physical properties of the four polymorphs of TiO2. Mol. Simul. 1991, 6, 239– 244. (34) Cornell, W. D.; Cieplak, P.; Bayly, C. I. A second generation force field for the simulation of proteins, nucleic acids, and organic molecules. J. Am. Chem. Soc. 1995, 117, 5179–5197. (35) Ryckaert, J. P.; Ciccotti, G. Numerial integration of the Cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J. Comput. Phys. 1977, 23, 327–341.
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10 A˚ and reaching zero at 12 A˚ was adopted, and the particleparticle particle-mesh (PPPM) solver was employed to calculate the long-rang electrostatic interactions. The lowest layer of TiO2 was frozen for the whole simulation time. Assemblies consisting of FN-III10 and TiO2 were initially energy minimized to remove bad steric contacts and then relaxed at constant temperature and volume over 100 ps with position restraints on the carboxyl oxygen atoms (OCOO-) to prepare the systems in such a state that unphysical forces did not cause improbable displacements. In the following 400 ps, the constraints were removed and FN-III10 began to move toward the surface. Finally, the resulting configuration of each system without any constraint was equilibrated for 4 ns. Every 4 ps a snapshot of the entire system was taken, enabling us to obtain a detailed analysis of the evolution of FN-III10 adsorption dynamics. Structural Descriptors. We aim to address the question of how much surface defects influence the adsorption properties of the FN-III10 molecule, with an understanding of the underlying adsorption mechanism of protein on the TiO2 substrates with various surface topographies. Hence, a set of global structural characteristic parameters, providing valuable insight into the overall packing stability of the protein on the microscopic level, were obtained to monitor the folding kinetics of FN-III10 during the simulation time. Radius of Gyration. As an indicator of structure compactness, the radius of gyration (Rg) of a single conformation for a protein molecule is defined as36 vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi u N u1 X Rg ¼ t jrj - Æræj2 ¼ N j¼1
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 u N N u1 X X 1 t ri rj N j¼1 N i¼1
ð1Þ
where ri and rj represent position vectors of the ith and jth atoms and N is the total number of atoms. Eccentricity. To obtain the eccentricity (η), it is necessary to introduce the moment of inertia matrix (I ) for a protein molecule, which can be calculated as follows37
(36) Huang, X. M.; Powers, R. Validity of using the radius of gyration as a restraint in NMR protein structure determination. J. Am. Chem. Soc. 2001, 123, 3834–3835. (37) Jefferson, F.; Raman, A. A relation between the principal axes of inertia and ligand binding. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 978–983.
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2
N X
Article
N X
mk ðyk þ zk Þ - ðyc þ zc Þ mk 6 6 k¼1 k¼1 6 6 6 NX N X 6 I ¼ 6 mk xk yk þ xc yc mk 6 k¼1 k¼1 6 6 6 N N X X 4 mk xk zk þ xc zc mk 2
2
2
2
k¼1
k¼1
-
N X
mk xk yk þ xc yc
k¼1 N X
Iave Imax
mk ðxk 2 þ zk 2 Þ - ðxc 2 þ zc 2 Þ
k¼1
-
N X
mk yk zk þ yc zc
k¼1
ð3Þ
μ ¼ D
qi ðri - rc Þ
ð4Þ
i¼1
where qi is the partial charge of the ith atom, N is the total number of atoms, ri and rc denote the position vectors of the ith atom and the center of charge, respectively, and D is a unit conversion factor, that is, D/(e 3 A˚). The angle between the unit vector normal to the TiO2 surface and the unit vector along the dipole of FN-III10 is referred to as the orientation angle whose cosine value can be used to represent the orientation of the adsorbed FN-III10 molecule. Root Mean Square Deviation. The root-mean-square deviation (rmsd) is a numerical measure of the difference between one simulated structure and its reference structure. The commonly used expression is vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi uN uP u ðri - ri, ref Þ2 t ð5Þ rmsd ¼ i ¼ 1 N where N is the total number of atoms in the selected segment and ri and ri,ref denote the instantaneous position vector and reference position vector of the ith atom, respectively. Normalized Distance. The displacements of O3 atoms in the top TiO2 layer during the simulation are extremely small because of their imperceptible interaction with FN-III10; therefore, the average of z coordinates of O3 atoms in the top layer is selected as the reference surface for all models. The normalized distance (dnor) from the center of mass of FN-III10 to the reference surface was calculated to monitor the approaching process of protein toward the TiO2 surface di dnor ¼ ð6Þ d0 where di is the instantaneous value of the distance from the center of mass of the protein to the reference surface and d0 is the initial value of di before energy minimization. Langmuir 2010, 26(20), 15972–15981
N X
N X
mk
k¼1
N X
mk xk zk þ xc zc
k¼1
-
mk
k¼1
where Imax is the maximal principal moment of inertia, Iave is the mean value of three principal moments of inertia, and Imin involved in later sections is the minimal principal moment of inertia. Dipole Moment and Orientation Angle. The dipole moment ( μ) can be obtained from N X
-
mk
k¼1
where xc, yc, and zc are the center-of-mass coordinates, xk, yk, and zk are the spatial data of the kth atom, mk is the mass of the kth atom, and N is the total number of atoms. Three eigenvalues of matrix I, which will certainly be real values, correspond to three principal moments of inertia. Hence, the eccentricity can be gained by η ¼ 1-
N X
N X
3 mk
k¼1
mk yk zk þ yc zc
k¼1 N X
N X
N X
mk
k¼1
mk ðxk 2 þ yk 2 Þ - ðxc 2 þ yc 2 Þ
k¼1
N X
mk
7 7 7 7 7 7 7 ð2Þ 7 7 7 7 5
k¼1
Bonding Interaction and Hydrogen Bond. According to Monti’s results,21 the average adsorption distance between Ti atoms and the adsorbate oxygens was about 2.10 ( 0.10 A˚, oscillating in the range of 1.80-2.60 A˚. Hence, in this article, it is considered to be a bonding interaction when the distances from the surface Ti atoms to the oxygen atoms of FN-III10 (OFN) are less than 2.5 A˚. Also, a pattern of hydrogen bonding (HB) that has already been used by many other authors38-41 is identified, which considers the donor-acceptor distance to be lower than 3.4 A˚ and the donor-H...acceptor angle to be larger than 135. Interaction Energy. In the following discussion, the timedependent interaction energy of the simulated systems was calculated according to EintðtÞ ¼ PTiO2 þ FN ðtÞ - PTiO2 ðtÞ - PFN ðtÞ
ð7Þ
where Eint(t) denotes the interaction energy between FN-III10 and the TiO2 surface at time t during the MD simulation, PTiO2þFN(t), PTiO2(t), and PFN(t) are the potential energies of the FNIII10-TiO2 complex, the TiO2 surface, and FN-III10 at time t, respectively.
III. Results and Discussions From the trajectory, it can be observed that a significant adsorption of FN-III10 on the rutile TiO2(110) surface occurred in all cases of MD simulations, accompanied by a certain conformational rearrangement of the interacting strands. The secondary structure of the protein, dominated by β strands, was affected to various degrees depending on the surface topology. Figure 3 gives the final states of four systems after the 4 ns equilibration stage in which the β strands are colored yellow and the RGD sequences protruding from the body of FN-III10 are shown in VDW mode. As compared with Figure 3b, the partial loss of secondary structures is a bit more obvious in Figure 3a, and the larger deformations of the protein displayed in Figure 3c,d may be due to the relatively larger contact areas. To understand the influence of surface topographies on the adsorption geometries of adsorbates, the FN-III10-TiO2 binding mode and the structural dynamics of FN-III10 during the simulation were analyzed in detail. FN-III10-TiO2 Binding Mode. The adsorption of protein onto the metal oxide layers was the result of several contributions from different types of interactions involving both side-chain and (38) Garcı´ a, A. E.; Stiller, L. Computation of the mean residence time of water in the hydration shells of biomolecules. J. Comput. Chem. 1993, 14, 1396–1406. (39) Obst, S.; Bradaczek, H. Molecular dynamics study of the structure and dynamics of the hydration shell of alkaline and alkaline-earth metal cations. J. Phys. Chem. 1996, 100, 15677–15687. (40) Song, D. P.; Chen, M. J.; Liang, Y. C.; Bai, Q. S. Adsorption of tripeptide RGD on rutile TiO2 nanotopography surface in aqueous solution. Acta Biomater. 2010, 6, 684–694. (41) Wu, C. Y.; Chen, M. J.; Guo, C. Q. Peptide-TiO2 interaction in aqueous solution: conformational dynamics of RGD using different water models. J. Phys. Chem. B 2010, 114, 4692–4701.
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Figure 3. Adsorption conformations of FN-III10 on rutile TiO2(110) surfaces after the 4 ns equilibration stage. (a) Model I, (b) model II, (c) model III, (d) model IV. FN-III10 is displayed by a new cartoon mode combined with a line mode, and TiO2 and RGD are shown by a line mode and VDW mode, respectively. The β strand, R helix, and 310 helix are highlighted in yellow, violet-red, and dark blue, respectively. The direction of the dipole moment of FN-III10 is indicated by a green arrow.
backbone atoms of FN-III10. RDFs of TiO2 surface atomsfunctional groups in FN-III10, the adsorbed sites, and the final conformations of adsorbed protein were introduced to determine the FN-III10-TiO2 binding mode. RDF Analysis. The RDF results of O (oxygens in carbonyl groups, hydroxyl groups and carboxyl groups)-Tis (surface titaniums), OOH (oxygens in hydroxyl groups)-Os (surface oxygens), and N (nitrogens in amide groups, guanido groups and NH3þ groups)-Os were obtained by averaging the instantaneous values of the final 400 ps of the MD equilibration stage of each assembly. As shown in Figure 4, the OCO (oxygens in carbonyl groups)Tis and OCOO- (oxygens in carboxyl groups)-Tis RDFs exhibit the first sharp peaks within the range of 2.00-2.10 A˚ in the four models without exception, indicating that several OCO and OCOO- atoms of FN-III10 are engaged in direct interactions with the available surfaces, in accordance with Monti’s conclusion42 that carboxyl and carbonyl groups were all possible coordination points of peptide on the rutile surface. Apart from these two groups, hydroxyl groups in model II-IV also interact with the TiO2 surface actively, as inferred from the OOH-Tis and OOH-Os RDFs. In model I, the distances from OOH atoms to Tis and Os atoms are far away from the defined bonding interaction distance and HB distance, respectively, and in models II-IV, the first sharp neighboring peaks of OOH-Tis RDFs are centered at 2.05 A˚, confirming that some OOH atoms bind to the surface Ti atoms successfully. Moreover, the presence of a low-amplitude OOH-Tis (42) Monti, S.; Carravetta, V.; Battocchio, C.; Iucci, G.; Polzonetti, G. Peptide/ TiO2 surface interaction: a theoretical and experimental study on the structure of adsorbed ALA-GLU and ALA-LYS. Langmuir 2008, 24, 3205–3214.
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RDF (approximate to 0) between the first two peaks indicates that the adsorbed hydroxyl groups have no tendency to be removed from the surface. The well-defined sharp peaks centered at 2.60-2.80 A˚ are clearly presented in the plots of OOH-Os RDFs; however, the formation of hydrogen bonds connecting the hydroxyl groups to the Os atoms mainly depends on the value of — OOHHOs. An appreciable but very small amplitude of Namide (nitrogens in amide groups)-Os RDF can be detected within the range of 2.55-3.40 A˚ in the four models, suggesting that the distances from some amide N atoms to the Os atoms are exactly within the HB range but that the number of involved Namide atoms is extremely small relative to the total number of Namide in FNIII10. The absence of an apparent sharp peak may be ascribed to the frequent interruption of these fairly weak hydrogen bonds. The Nguanido (nitrogens in guanido groups)-Os RDFs of models I-II show the first sharp peak at ∼2.85 and ∼2.80 A˚, respectively, indicating that some guanido groups approach the surface O atoms at HB distances whereas the distances from Nguanido of model III and NNH3þ of models I-II to Os are far away from the suggested HB distances. The guanido groups in model IV and the NH3þ groups in model III-IV, without exception, are all out of the neighborhood of the TiO2 surface (r e 10 A˚). Adsorbed Groups. According to the RDF results, it can be inferred that multiple coordinations together with hydrogen bonding determine the strong protein-surface complexes. To examine the FN-III10-TiO2 interaction in more depth, information about newly formed bonds that connect FN-III10 to the substrate surface has been summarized in Table 2 by averaging the values of the last 1.2 ns equilibration stage of each model. Langmuir 2010, 26(20), 15972–15981
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Figure 4. RDFs of TiO2 surface atoms-functional groups in the FN-III10 molecule. (a) Model I, (b) model II, (c) model III, and (d) model IV. Table 2. Types of Adsorbed Groups, Numbers of Newly Formed Bonding Interactions, Hydrogen Bonds, and Involved Residues
a
Average number of newly formed Ti-OFN bonding interactions. b Average number of newly formed hydrogen bonds. c Average number of involved residues.
In model I, the most adjacent groups to the TiO2 surface were the positively charged guanido group in Arg93, carbonyl groups in Thr39, Gly40, 41, 65, and Asn42, the amide group in Gly40, and negatively charged carboxyl groups in Asp67 and Ile96. From the trajectory, it can be seen clearly that the adsorbed charged groups with larger initial separations from the surface (>18 A˚) moved toward the surface more quickly than the mentioned carbonyl groups. The FN-III10 molecule readjusted its structure constantly in the early stage of adsorption, which may originate from the eagerness of the protein for a more favorable configuration to maximize the interaction with the metal oxide layer; however, the number of β strands remained unchanged until the end of the simulation, but their lengths were shortened to varied Langmuir 2010, 26(20), 15972–15981
extents with increased proportions of turns and coils. A relatively rich variety of adsorbed groups can be observed in model II, including the positively charged guanido group in Arg93, amide groups in Asn42 and Ile96, carbonyl groups in Thr39, Gly40, 41, 65, Asn42, and Pro64, the hydroxyl group in Thr39, and negatively charged carboxyl groups in Asp67, Glu95, and Ile96. The carbonyl O atoms in Gly40 and Pro64 were substituted into two O vacancies in a stable adsorbate configuration, and the remaining O vacancies remained unoccupied because they were outside the protein-surface contact area. The secondary structure of protein was well reserved, except that β strand A split into coils and turns because of the local arrangement of the residues around it. DOI: 10.1021/la103010c
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Wu et al. Table 3. Averaged Properties of the Adsorbed FN-III10
parameters
Rg (A˚)
η
Imax/Imin
μ (D)
cos θ
dnor
crystal model I model II model III model IV
13.41 12.29 ( 0.06 12.91 ( 0.09 13.40 ( 0.06 14.39 ( 0.10
0.237 0.190 ( 0.005 0.233 ( 0.004 0.189 ( 0.004 0.215 ( 0.003
2.54 1.98 ( 0.04 2.18 ( 0.04 1.48 ( 0.03 1.86 ( 0.03
346.11 198.54 ( 6.17 306.98 ( 6.34 711.00 ( 11.94 788.58 ( 7.07
0.82 0.76 1.00 1.00
0.625 ( 0.009 0.655 ( 0.008 0.410 ( 0.004 0.627 ( 0.006
The existence of a step and groove in models III and IV weakened the interatomic interaction of TiO2 atoms around the edges, leaving many unstable undercoordinated atoms that deviated from their initial positions after relaxation, resulting in increased surface energy. As Baier stated,43 the higher surface energy led to a more stable adsorption and a more extensive spreading. Model III ranked first in the average number of Ti-OFN bonding interactions (nBI) and hydrogen bonds (nHB), but the secondary structure of the adsorbed FN-III10 was dominated by floppy coils, with only shortened β strands D and E retained. More than 20 atoms in FN-III10 were attached to the step inner TiO2 atoms, among which OCOO- in Asp67 and OCO in Gly37 and Val66 formed a tridentate coordination with one Ti atom located at the bottom of the step. Also, the carbonyl oxygens in Tyr92 and Arg93 and the hydroxyl oxygen in Tyr92 adsorbed on the step edge along the y direction explicitly, and the hydroxyl group in Ser81 and the carbonyl and carboxyl groups in Asp80 (contained in the RGD sequence) formed direct interactions with the Ti atoms on the step edge along the x direction. However, the adsorption of RGD highly restricted its movement and thereby may decrease the biological activity of this sequence. As for model IV, the amide group in Gln46, carbonyl groups in Gly41 and Gln46, and carboxyl groups in Glu95 and Ile96 were involved in direct interactions with TiO2 atoms on the groove edges. As shown in Figure 3d, the β strands were considerably disrupted, with only a portion of β strands D and F unchanged. β strands A and G were converted to an R helix, and the coil composed of Thr28-Arg30 transformed into a 310 helix. The massive modification of secondary structure were mainly derived from the excessive stretch of the FN-III10 molecule to reach the groove edges, which had a high reactivity and a great affinity for the model protein. Structural Dynamics of FN-III10. Once the protein adsorbed onto the TiO2 surface, its structure would be gradually adjusted to the optimized conformation to maximize the adsorbate-adsorbent interaction. A set of global structural parameters to characterize the conformation of FN-III10, including the radius of gyration (Rg), the ratio of the maximal to the minimal principal moment of inertia (Imax/Imin), the eccentricity (η), the dipole moment ( μ), the orientation angle (cos θ), the normalized distance from the center of mass of the protein to the reference surface (dnor), and the rmsd of RGD, were adopted to analyze the structural stability of the protein on different rutile TiO2(110) surfaces. The reported results in Table 3 were averaged over the last 1.2 ns equilibration stage. Orientation Distribution of FN-III10. From Table 3 and Figure 5, it can be observed that the values of cos θ for FNIII10 in model III-IV approach 1.00 perfectly with an extremely narrow orientation distribution and that the directions of dipole moments (green arrows in Figure 3) are nearly parallel to the unit normal vector of the surface. The dipole moment of FN-III10 on the perfect TiO2 surface (198.54 D) is much smaller than that in its (43) Baier, R. E.; Meyer, A. E.; Natiella, J. R.; Natiella, R. R. Surface properties determine bioadhesive outcomes: methods and results. J. Biomed. Mater. Res. 1984, 18, 327–382.
15978 DOI: 10.1021/la103010c
native state (346.11 D), and the corresponding parameters of FNIII10 on the step and groove surfaces are 2.05 and 2.28 times larger than the native dipole moment as a result of drastic structural rearrangement induced by more highly active adsorption sites provided by the step or groove. Although a larger dipole moment favors a narrower orientation distribution, severe conformational changes in FN-III10 from its native structure on the latter two surfaces may result in the denaturation of the protein. The orientation angles of FN-III10 in model II, by contrast, disperse a little widely with a dipole moment (306.98 D) that is close to the starting value of the protein, indicating that the adsorption conformation of the adsorbate does not deviate from its native structure but adjusts within a small fluctuation range. A detailed analysis of the conformational information will be presented below. Shape of FN-III10. The radii of gyration of FN-III10 in models II and III (Figure 6) lie within the range of 12.63-13.58 A˚, among which the average value of model III during the later stage (13.40 ( 0.06 A˚) is in close proximity to the value of the starting FN-III10 conformation (13.41 A˚). However, the V-shaped distribution of Rg during the first 200 ps implies that FN-III10 greatly readjusts itself when approaching the step surface, but the marked oscillation of Rg declines rapidly after adsorption. Thus, it is reasonable to believe that although the value of Rg is equal to the initial structure of FN-III10, the protein in model III displayed a modified but stable conformation (Figure 3c). The Rg of FNIII10 in models I and IV displays two different trends: the former gradually decreases from the initial value to about 12.29 A˚ with β strands shortened considerably, and the latter increases to about 14.39 A˚, indicating a lower packing density of the protein. The eccentricity (Figure 7) and Imax/Imin (Figure 8) are also commonly used parameters in characterizing the overall shape of a protein. The starting Imax/Imin and η of FN-III10 are greater than 1 (2.54) and zero (0.237), respectively, indicating an initially elongated shape that is approximately cylindrical. The adsorbed FN-III10 molecules in models I, III, and IV look more globular as evidenced by lower values of Imax/Imin and η. (For model I, Imax/ Imin = 1.98 ( 0.04, η = 0.190 ( 0.005; for model III, Imax/Imin = 1.48 ( 0.03, η = 0.189 ( 0.004; for model IV, Imax/Imin = 1.86 ( 0.03, η = 0.215 ( 0.003). Especially in model III the Imax/Imin value falls sharply during the first 400 ps and then the oscillation around the valley (1.39) continues for about 1 ns, after which a slight turning to 1.41 can be detected, and Imax/Imin increases steadily over the final 1.4 ns as time progresses. The time division of conformational change is also reflected in the plot of η, which exhibits three step changes following a steep decrease. In model II, the Imax/Imin value turns abruptly at about 1.8 ns because of the reorientation of some β strands for a more favorable configuration, especially β strand A, which splits into coils and turns at that moment. During the last 1.2 ns, Imax/Imin and η explored in model II center on 2.18 and 0.233, respectively, which are close to those of the native conformation. The normalized distances (dnor) from the center of mass of FNIII10 (green dots in Figure 3) to the respective reference surface, displayed in Figure 9, decline rapidly during the deposition stage but gradually shift to a gentle phase, except in model II, where dnor Langmuir 2010, 26(20), 15972–15981
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Figure 5. Orientation distribution of FN-III10 adsorbed on the adopted TiO2 surfaces. Figure 8. Imax/Imin as a function of the simulation time for the FN-III10 molecule.
Figure 6. Radius of gyration as a function of the simulation time for the FN-III10 molecule.
Figure 9. Normalized distance from the center of mass of FN-III10 to the reference surface as a function of the simulation time.
Figure 7. Eccentricity as a function of the simulation time for the FN-III10 molecule.
undergoes a more marked fluctuation during the first 1.8 ps as a result of the constant structural readjustment of FN-III10. The values of dnor in the first two models finally converge to 0.625 and 0.655, respectively, and one of the factors leading to two similar normalized distances is the same elevation of the settling planes. As for model III, FN-III10 is at the bottom of the step, thus a smaller dnor (0.410) is presented, and the dnor for model IV (0.627) Langmuir 2010, 26(20), 15972–15981
Figure 10. Adsorbed structure of FN-III10 (red) in (a) model I, (b) model II, (c) model III, and (d) model IV superimposed on the crystal structure of FN-III10 (dark blue).
is much larger, even though the depth of the groove is twice as large as the step. In model IV, seven residues next to the groove bottom interacted with the TiO2 atoms directly, but the DOI: 10.1021/la103010c
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Figure 11. rmsd change in a single residue in an RGD sequence without H atoms as a function of the simulation time. (a) Model I, (b) model II, (c) model III, and (d) model IV.
unattached portions of FN-III10, especially the RGD sequence, tried to stretch along the direction normal to the surface because of the decreased influence of the surface atoms and the high flexibility of the Arg side chain. Hence, the center-of-mass position was lifted because of the elongated FN-III10 molecule with its long axis, and the protein lengthened along the short axis at the same time, resulting from a larger contact surface. Therefore, the adsorbed FN-III10 in model IV exhibited a loose conformation with a larger Rg and Imax/Imin than that of model III, as listed in Table 3. To assess the conformational change of the adsorbed protein visually, the structures of FN-III10 on different surfaces were imposed on its crystal structure by the VMD software. From Figure 10, it can be seen that the structural features of the proteins in models I and II are preserved to a large extent in comparison with its crystal structure, and a high degree of structural deformation occurs after FN-III10 adsorption on the surfaces with a step or groove. The interaction energies for models I-IV are -434.13, -747.11, -1534.98, and -1126.75 kcal 3 mol-1, respectively. Thus, the high surface energy can be viewed as a positive ingredient in helping to promote a stable protein adsorption, but too strong an interaction may cause a severe conformational deformation and thereby the denaturation of the adsorbed protein. Conformational Changes of RGD. The RGD sequence in FN-III10 was known to serve as a primary cell-attachment cue and modulate cell adhesion through the interactions with integrin receptors. Much research has been carried out to investigate the 15980 DOI: 10.1021/la103010c
responsibility of RGD in cell attachment,6,41,44,45 which is critical for material colonization and thereby has its full impact on the biocompatibility of the materials. Terminated with a positively charged residue (Arg) and a negatively charged residue (Asp), RGD has considerable structural flexibility. Therefore, the adsorption behavior of FN-III10 may have a strong influence on the conformation of RGD, thus affecting the cellular response induced by this sequence. The evolution of rmsd for residues contained in RGD without H atoms during the final 4 ns adsorption stage is presented in Figure 11, in which the structure after energy minimization is selected as the reference. The Arg residue exhibits the most flexible conformation because of a long side chain and deviates from the reference structure greatly, and the rmsd of the Asp residue is always the smallest one in all cases. The rmsd curves in model III fluctuate more mildly than the corresponding ones in the other three models. As Figure 11c shows, the mobility of the Asp residue was highly restricted once it adsorbed onto the step edge, but the rmsd of Asp turns abruptly at 1 ns because of the eagerness for more bonding interactions with the TiO2 surface and hovers around 27.02 and 28.10 A˚ in these two successive time intervals, respectively. The adsorption conformations of RGD in model III are amplified in Figure 12 for a better visual effect. Two OCOO- atoms of Asp are adsorbed on (44) Ruoslahti, E.; Pierschbacher, M. D. New perspectives in cell adhesion: RGD and integrins. Science 1987, 238, 491–497. (45) Chen, M. J.; Wu, C. Y.; Song, D. P.; Li, Kai. RGD tripeptide onto perfect and grooved rutile surfaces in aqueous solution: adsorption behaviors and dynamics. Phys. Chem. Chem. Phys. 2010, 12, 406–415.
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Figure 12. Representations of the adsorbed RGD sequence in model III at different times by MS. (a) 920 and (b) 1080 ps. TiO2 and the Asp residue of RGD are displayed by the CPK mode and the ball-and-stick mode, respectively. The Arg and Gly residues of RGD are represented by the stick mode, and the remainder of FN-III10 is shown by the line mode.
two adjacent Ti atoms at 920 ps, but one of the OCOO- atoms moved downward to form a bidentate coordination with two Ti atoms after 1 ns and the OCO also reoriented itself to bond to an undercoordinated Ti atom, as a result of the high affinity of the coordinatively unsaturated Ti atoms to carboxyl and carbonyl groups.
IV. Conclusions The FN-III10 molecules were placed close on the perfect and three reduced rutile TiO2(110) surfaces with different types of defects in vacuum to inspect the special effect of surface defects on the adsorption behavior of the model protein on a 4 ns timescale. Significant adsorption of FN-III10 on the TiO2 surface occurred in all cases of MD simulations. Multiple coordination together with hydrogen bonding, involving both side-chain and backbone atoms, determined the strong protein-surface complexes. The analysis for the overall packing stability of FN-III10 in four different systems revealed that the adsorption affinity and conformational change in the protein largely depended on the surface topology. The adsorbed FN-III10 on the perfect surface exhibited a less elongated shape with seven shortened β strands as a result of peptide anchoring, and the reduced surfaces were shown to provide additional adsorption sites with higher activity. Two O vacancies in model II were substituted by OCO atoms in a stable adsorbate configuration, and the secondary structure of the model protein was well reserved except for the collapse of β strand A. A high degree of structural deformation was presented after FN-III10 was adsorbed on the surfaces with a step or groove. Model III ranked first in the interaction energy with a modified configuration, dominated by floppy coils, but the adsorption of
Langmuir 2010, 26(20), 15972–15981
RGD highly restricted its movement and thereby may decrease the corresponding biological activity. In model IV, the protein exhibited a lower packing density, and a portion of the structure was converted to the R helix and 310 helix because of the excessive stretch of FN-III10 to reach the groove edges. A special orientation of the adsorbed protein;almost perpendicular to the TiO2 surface with an extremely narrow distribution;was presented on the step and groove surfaces. Thus, high surface energy can be viewed as a positive ingredient in helping to promote stable protein adsorption, but too strong of an interaction may cause a severe conformational deformation and thereby the loss of bioactivity of the adsorbed protein, which is not favorable for practical applications. The present work can be considered to be a positive report on the structural dynamics of the adsorbed protein on the microscopic level by means of MD simulations. Although 4 ns is a relatively short duration over which to obtain a completed exploration of the protein-TiO2 adsorption mechanism, the modeling results suggests that it will be very interesting to design a patterned surface to ensure both the native conformation of the immobilized FN-III10 molecule and its strong interaction with the substrate surface. Investigations of the adsorption dynamics of FN-III10 on the rutile (110) surfaces with more complicated topographies in aqueous solution, which is the final goal of our study, have already been undertaken. Acknowledgment. We acknowledge the New Century Elitist Supporting Program Foundation of the Ministry of Education of China (no. NCET-06-0332) and the 111 project (no. B07018) for financial support.
DOI: 10.1021/la103010c
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