Surface-Dependent Mechanical Stability of Adsorbed Human Plasma

Article pubs.acs.org/Langmuir

Surface-Dependent Mechanical Stability of Adsorbed Human Plasma Fibronectin on Ti6Al4V: Domain Unfolding and Stepwise Unraveling of Single Compact Molecules Virginia Vadillo-Rodríguez,* José M. Bruque, Amparo M. Gallardo-Moreno, and M. Luisa González-Martín Department of Applied Physics and Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), University of Extremadura, Avda de Elvas s/n, 06006 Badajoz, Spain ABSTRACT: In this study, the structure and mechanical stability of human plasma fibronectin (HFN), a major protein component of blood plasma, have been evaluated in detail upon adsorption on the nonirradiated and irradiated Ti6Al4V material through the use of atomic force microscopy. The results indicated that the material surface changes occurring after the irradiation process reduce the disulfide bonds that typically preclude the mechanical denaturation of individual HFN domains and interfere significantly with the intraionic interactions stabilizing the compact conformation of the adsorbed HFN molecules. In particular, upon adsorption on this material, the molecules adopt a more flexible conformation and become mechanically more compliant. Unexpected observations also indicated that, regardless the material surface, a single HFN molecule can be pulled into an extended conformation without the unfolding of its domains through a series of three unraveling steps. The forces involved in the unraveling process were found to be generally lower than the forces required to unfold the individual protein domains. This report is the first one to present the force displacement details associated to the straightening of a single compact protein at the molecular level.

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INTRODUCTION Understanding how proteins adsorb at solid−liquid interfaces is of increasing interest because of its implications for safety in a broad range of biomedical, industrial and technological applications.1,2 In medical applications, for instance, the adsorption of serum and tissue proteins on synthetic biomaterials used for artificial organs, vascular or orthopedic prostheses can lead to severe and recurrent infections due to the specific adhesion of bacterial cells to the protein-coated biomaterials.3,4 In the case of Ti6Al4V, the most common titanium alloy used in dental and orthopedic implants, the interaction with proteins is most likely influenced by a thin titanium oxide layer that spontaneously forms on the surface of this alloy. Importantly, this layer has been recently shown to confer to Ti6Al4V extraordinary bactericidal properties in vitro following its irradiation with UV−C light.5,6 The origin of this phenomenon has been attributed to the surface emission of radiation and small surface currents that occur as a consequence of the electron−hole pair recombination that takes place after the excitation process. The influence of these surface changes on the adsorption of proteins, a process that always precedes bacterial adhesion to any implanted material surface, has not yet been elucidated at the molecular level. Human plasma fibronectin (HFN), a large and essential glycoprotein that exists in soluble form in body fluids and in an insoluble from in the extracellular matrix, plays a major role in © XXXX American Chemical Society

the interaction of synthetic implants with the surrounding matrices. Typically, it functions as a key link between cells and their extracellular matrices and is now recognized to be the target for a large number of bacterial surface proteins as well.7,8 HFN is secreted as a dimer, with a monomer molecular weight of ∼220−250 kDa. The monomers, each 60−90 nm in length and 2−3 nm in thickness, are connected via two flexible disulfide bonds near their carboxyl termini. Each monomer consists of three structurally homologous repeating modules that are arranged like a string of beads connected by short polypeptide segments.9,10 The three modules, referred as FnI, FnII, and FnIII, are globular, and, separately, X-ray and NMR studies have revealed that they are all constructed of antiparallel β-sheets.11−13 These sheets interact through hydrogen bonding that considerably stabilizes the modules; FnI and FnII are further stabilized by a pair of disulfide bonds occurring between particular β-strands within them.1,14 As for the tertiary structure of the entire protein, no success in its crystallization has been reported until now, but studies on proteolytic and recombinant protein fragments have indicated that the compact conformation of soluble HFN is stabilized by intermolecular ionic interactions between the two monomers.15 Received: March 13, 2013 Revised: June 17, 2013

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Figure 1. Representative AFM height images recorded in contact mode for the bare nonirradiated (a) and HFN molecules adsorbed on nonirradiated (b) and irradiated (c) Ti6Al4V samples under PBS solution. The white boxes in (c) highlight individual globules. The images correspond to a sample area of 2 × 2 μm2 (z-range: 6.8 nm). deionized water (Milli-Q system), 70% acetone, and ethanol for periods of 10 min each. Finally, they were dried in an oven at 40 °C for 1 h and stored in a desiccator for no longer than 24 h. A set of these samples were exposed to an UV−C source for 15 h. G15-T8 UV lamps, emitting predominantly at a wavelength of 254 nm, were kindly provided by Philips Iberica. The disks were positioned at 10 cm from the light source and centered, receiving an intensity of about 4.2 mW cm−2. The irradiation installation was inside an opaque wood chamber to prevent interference from the room or daylight, or prevent any damage to the users. Human plasma fibronectin (HFN) was obtained from Sigma Aldrich (0.1% w/v) and used without further purification. The protein was suspended in phosphate buffered saline (PBS) to concentrations of 1, 10, 50, 100, and 200 μg/mL and stored at 4 °C in plastic vials prior to use. To coat the Ti6Al4V surfaces with HFN, sterile silicone chambers (FlexiPERM, Greiner Bio-One), fixed to the disks surfaces by applying a small vertical pressure, were used. These chambers were filled with 0.5 mL of protein solution and subjected to slight orbital shaking. After 15 min, the silicon chambers were carefully removed and the HFN coated disks rinsed with PBS to remove loosely bound proteins. The irradiated samples were immediately coated with HFN once the irradiation process was completed. Following the 15 min of adsorption time, the coated irradiated substrata were transferred to the AFM and studied for up to 1 h. After this time, the samples were disregarded and replaced for freshly prepared new samples. Atomic Force Microscopy. Experiments were performed in PBS using an Agilent AFM 5500 (Agilent Technologies) operating at room temperature and rectangular silicon cantilevers with a nominal spring constant of 0.03 N m−1 and a specified tip radius of ∼8 nm (CSC38 series, MicroMash). Prior to use, the AFM piezoelectric scanner was calibrated to ensure scanner accuracy in the x, y, and z directions using two certified silicon calibration gratings (TGX01and TGZ01) from MikroMasch. The spring constant of the cantilevers was individually determined by means of the thermal noise technique provided with the instrument software. Prior to experiments, the different protein concentrations were tested to find the best experimental conditions for AFM experimentation. It was found that 100 μg/mL produced a high probability (∼2−3%) of single molecule attachments. At this concentration, three to five independent samples were studied for each experimental condition. For each sample, three topographical images and hundreds of force−distance curves were collected at randomly selected locations. AFM topographical images were recorded in contact mode with an applied force maintained below 1 nN at a scan rate of 2 Hz. The adsorbed protein layers were found to remain stable and no protein detachment was observed upon repeated scanning in any of the conditions investigated. Force−distance curves were collected at a constant loading force of 1 nN and Z piezo velocity of 1 μm s−1. Regardless of the material employed for protein adsorption, a fraction of about ∼5−10% of the recorded curves displaying specific molecular events showed force transitions occurring in a stepwise fashion. A data set was considered to be complete when at least 30 force plots displaying specific molecular events (i.e., with a sawtooth or stepwise pattern) were acquired. These curves were used to generate the histograms presented. At the end of each experiment, force−distance curves were also collected on clean glass surfaces to

It has been proposed that, in addition to chemical cues, mechanical forces play a critical role in regulating the functional states of HFN. The binding sites buried within the protein in its native state, also referred as cryptic sites, might be potentially exposed through mechanical stretching.16,17 Assessing the mechanical stability of proteins has been made experimentally possible through the use of atomic force microscopy (AFM), a nanotool capable of stretching single molecules. The first protein to be subjected to AFM mechanical unfolding was the giant muscle protein titin.18,19 Since then, a few other modular proteins, such as tenascin,20 spectrin,21 fibronectin,22 ubiquitin,23 or fibrinogen,24 have been probed by mechanical force in a similar manner, although most studies have made use of artificially constructed homo- and heteropolymers to circumvent the intrinsic heterogeneity of the natural modular proteins.25−29 Collectively, these investigations have shown that the individual domains of modular proteins behave like elements of a linearly jointed spring, and unfold abruptly at a critical force that range between 50 and 300 pN. Accordingly, it is typically observed that the forced unfolding of a modular protein exhibit a multipeaked force-displacement pattern. In addition to measuring inter- and intramolecular interactions, AFM has proven to be a valuable tool to image single molecules in situ under physiological conditions. This AFM capability has been used to investigate the structure of adsorbed proteins on various material surfaces.30,31 HFN, for instance, has been found to adopt an extended conformation on mica and silica,10,32 but was found to adsorb in globular form on titanium materials.33,34 The surface dependent conformation of HFN on Ti6Al4V have not yet been addressed. The aim of this work is to assess the influence of the Ti6Al4V surface properties, prior to and after subjecting the material to the UV−C radiation process, on the adsorption of HFN. The conformation and mechanical stability of the adsorbed molecules are evaluated under physiological buffer through the combined use of AFM imaging and force spectroscopy with molecular resolution. The results obtained are discussed in terms of the structural properties of the adsorbed molecules, which are shown to depend largely on the surface properties of the material.

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MATERIALS AND METHODS

Sample Preparation. Disks of Ti6Al4V were cut from bars of 25 mm in diameter supplied by DKSH Switzerland Ltd., a world leader provider of titanium alloys for orthopedic and medical applications. The disks were abraded on successively finer silicon carbide papers, mechanically polished with diamond paste, and finished with colloidal silica. Prior to their use, the Ti6Al4V disks were carefully cleaned with DSF disinfectant (DERQUIM DSF 11; Panreac Quimica S.A.) and distilled water at 60 °C by vigorously rubbing with a smooth cotton cloth and then rinsed repeatedly with distilled water and sonicated in B

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Figure 2. Typical AFM force-plot obtained upon stretching individual HFN molecules adsorbed on the nonirradiated Ti6Al4V material (a), together with the corresponding histograms of the measured unfolding forces (b) and lengths (c). (d−f) Equivalent plots obtained upon stretching individual HFN molecules from the irradiated Ti6Al4V material. The distance between the dashed lines in (a) and (d) between consecutive peaks represents the measured unfolding lengths (for example, see black arrows). The solids lines in the histograms are Gaussian fits to the data. check for tip contamination or damage. All mean values reported in this paper are given with a standard deviation.

white boxes), which display similar morphology to those seen on the nonirradiated samples. It clearly appears that the small forces occurring between the AFM tip and the sample are sufficient to unravel the soft protein globules adsorbed on the irradiated surfaces. Apparently, upon adsorption on the irradiated material the intramolecular forces that could hold the molecule in a relative compact conformation have been weaken or partially disrupted. XPS analysis revealed that there were no significant differences in the elemental chemical composition of the nonirradiated and irradiated material surfaces (data not shown). Similarly, streaming potential measurements showed that the UV irradiation of the Ti6Al4V material did not produced significant changes in the values of the measured zeta potentials.35 However, water contact angle measurements demonstrated that the initially hydrophobic nonirradiated Ti6Al4V material became highly hydrophilic after the irradiation process.35 As a general rule, proteins undergo greater structural changes upon adsorption to hydrophobic than hydrophilic surfaces.36 Dehydration, a process related to the release of a large number of water molecules during adsorption, promotes such structural changes. In contrast, it has here been detected that HFN experiences a greater structural change upon adsorption to the hydrophilic (irradiated) than hydrophobic (nonirradiated) Ti6Al4V material. Interestingly, it has been recently shown that treating the Ti6Al4V material with UV−C light provokes a postresidual surface emission that likely takes place within the UV wavelength range.5 In this context, a few latest studies have also shown that UV−C light is able to break disulfide bonds in plasma proteins via photolysis.37,38 Since many of the HFN modules are stabilized by disulfide bridges, it is thus expected that these bonds may be disrupted by the surface emission of UV radiation, conferring to the molecules a more flexible and looser conformation.

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RESULTS AND DISCUSSION AFM Topographical Images. Figure 1 shows representative AFM height images recorded in contact mode for the bare nonirradiated (a) and HFN molecules adsorbed on nonirradiated (b) and irradiated (c) Ti6Al4V samples under PBS solution. These images were collected with the same AFM tip and equal tip surface forces (∼0.1 nN). The irradiated material showed the same topographical characteristic as the nonirradiated material, i.e. a flat and featureless surface. The measured root-mean-square roughness, RMS, of the bare (nonirradiated and irradiated) substrata amounted to 0.28 ± 0.07 nm for the scanned area of 2 × 2 μm2. As can be seen in Figure 1b, individual HFN globules are present over the entire nonirradiated material, with a few aggregates also visible. Crosssectional analysis of the individual globules (n = 10) revealed an average width and height of 58 ± 6 nm and 2.8 ± 0.8 nm, respectively. Adsorbed HFN in globular form has also been observed by Sousa et al. on TiO2 surfaces for which a mean width of 55 ± 9 nm and height of 4.6 ± 1.6 nm were measured.33 MacDonald et al. found on pure Ti HFN globules with an average length of 16.5 ± 1, a height of 2.5 ± 0.5 nm and a width of 9.6 ± 1.2 nm.34 Elongated HFN molecules have also been imaged on surfaces such as mica and silica with an end to end distance that varies between 110 and 160 nm.10,32 The reported variability on the dimensions for the adsorbed HFN molecules can be attributed to the different substrata and solution conditions employed in the different studies. Figure 1c shows that the HFN protein layer adsorbed on the irradiated surface presents stretchable fibril-like structures; the orientation of these fibril-like features were depending on the scanning direction. A few globules were also detected (highlighted by the C

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Figure 3. AFM force-plots (a) obtained upon unraveling adsorbed HFN molecules (without the unfolding of its domains) from the nonirradiated and irradiated Ti6Al4V material displaying one, two, and three steps. Dashed arrows represent the total extension length associated to each curve. Black arrows are a representative example illustrating how the step force and length have been measured for each particular step displayed on the curves. (b) Histograms showing the distribution of the total extension length and (c) step force-step length relationship obtained from the recorded force-plots upon unraveling the HFN molecules from the different material surfaces. Solid lines in (c) represent the exponential fit to the data.

AFM Force Spectroscopy: Protein Domain Unfolding. A typical force plot obtained upon stretching an HFN molecule from the nonirradiated material is shown in Figure 2a. As it is widely understood now, the sawtooth pattern observed corresponds to the sequential unfolding in an all-or-none fashion of individual protein domains. The absolute value of the peak force relates to the mechanical stability of the domain, and the distance to the following peak to the length of the previously unraveled domain.39 Sets of these curves were used to generate histograms of the measured unfolding forces and corresponding lengths (Figure 2b and c). As can be seen in Figure 2b and c, the large majority of the unfolding events show a mean unfolding force of 112 ± 24 pN and length of 25.6 ± 3.7 nm. These results are similar to those reported by other authors studying the unraveling of native fibronectin through AFM.22,40,41 The measured values are attributed to the unfolding of the FnIII domains, while the contribution to the curves of the FnI and FnII modules is thought to be hampered by the intramolecular disulfide bonds that stabilize these domains, preventing their unfolding under usual experimental conditions. However, experimental and molecular simulation studies performed with artificially constructed homopolymers comprising copies of single FnIII modules have found that the unfolding of these domains occurs through several intermediate states; these intermediate states have not been detected in our measurements.22,42−44 It appears likely that the mechanical properties of single domains are not strictly given their amino acid sequence, but that can be regulated by the local environment and the interaction with the surrounding molecules. Figure 2d shows a representative force plot obtained during the mechanical stretching of an HFN molecule adsorbed on the

irradiated Ti6Al4V material. In this case, different types of unfolding events are distinguished in a single force plot. The obtained histograms reveal typical unfolding forces that range between 30 and 230 pN (Figure 2e) and three main classes of unfolding lengths (Figure 2f). Gaussian fits to the experimental data show a mean force of 107 ± 37 pN and mean lengths of 13.5 ± 3.2, 18.1 ± 1.1, and 26.9 ± 4.8 nm. As discussed above, the disulfide bonds present within the FnI and FnII modules have likely been reduced upon protein adsorption on the irradiated material. Based on this, the distinct unfolding events could be attributed to the unraveling of the FnI, FnII, and FnIII domains, for which natural occurring lengths at full extension are 17.1, 22.8, and 34.2 nm, respectively. Interestingly, the area enclosed by the Gaussian peaks (corresponding to the number of unfolding events for each particular length; Figure 2f) expressed as a percentage, that is, 26%, 6%, and 68%, is in relatively good agreement with the natural occurrence of FnI, FnII, and FnIII in human plasma fibronectin: FnI, FnII, and FnIII occur 12, 2, and 15 times, equivalent to 38%, 6%, and 56% of all domains in native HFN.14 The Ig-type domains of several cell adhesion molecules containing disulfide bonds buried in their core have also been found to fully extent under an AFM tip in the presence of reducing agents such as dithiothreitol.45,46 The addition of the reducing agents not only allowed full extension of the molecules but tended to increase the number of observed unfolding events. Similarly, under our experimental conditions, the average number of unfolding events was 5 ± 3 and 9 ± 5 for the HFN molecules adsorbed on the nonirradiated and irradiated Ti6Al4V, respectively. AFM Force Spectroscopy: Protein Unraveling. A small but significant fraction of the recorded force plots were found to display unexpected force transitions occurring in a stepwise D

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Figure 4. Model illustrating the force-induced straightening of an initial compact HFN molecule without the unfolding of its domains. HFN under physiological conditions assumes a compact conformation stabilized through electrostatic contacts between the FnIII 2−3 domains of one monomer and the FnIII 12−14 domains of the other monomer (a). It is hypothesized that an external force would first induce the extension of one of the Nterminal region (b), after which it would provoke the consecutive breakage of the two electrostatic contacts (indicated with black arrows; c and d). The folded diameter of the each module type has been reported to amount to 2.5 nm for FnI, 0.7 nm for FnII, and 3.2 nm for FnIII.53−55 Thus, the end-to-end length of a totally extended molecule is about ∼160 nm.

other monomer.15 If this connectivity prevails upon adsorption, then the extension from the compact to the extended conformation could occur through a series of three unraveling steps as illustrated in Figure 4. The first one would involve the extension of one of the N-terminal region (Figure 4b), and the two that proceed would correspond with the consecutive breakage of the two electrostatic contacts (Figure 4c and d). Because the AFM tip picks up the protein at a random location along its length, the total number of recorded plateaus varies from one to three, while the total measured length was typically found to range between 20 and 180 nm (Figure 3b). Moreover, as seen in Figure 3c, the unraveling forces, which result from the reduction of the number of possible molecular configurations upon stretching and hence, are of entropic origin, were found, as expected, to be generally lower than the forces require to break the multiple hydrogen bonds that hold together the HFN domains.56,57 Figure 3c also shows that, regardless of the material surface upon which the molecules were adsorbed, these forces are high if the disruption involves little extension, and lower if the forces that maintain the compact conformation are distributed over a longer distance. Particularly, it is found that length of the plateaus exponentially increases as the step force associated to each plateau decreases. The calculated exponential decay lengths were of 10 ± 2 and 30 ± 7 nm for the HFN molecules absorbed on the nonirradiated and irradiated Ti6Al4V material, respectively. These results indicate that for the same extension range the breaking of the ionic interactions stabilizing the adsorbed compact molecules occurs at a lower force on the irradiated than on the nonirradiated material. It is likely that the emission of UV radiation or the small surface currents occurring after the material is irradiated interfere with the intraionic interactions that stabilize the HFN molecules, rendering them more flexible against unraveling upon adsorption on this material. Finally, it was also observed that the molecules adsorbed on the irradiated material could occasionally be entirely stretch in a single step (Figure 3c), indicating that the pair of electrostatic contacts that holds the molecules in a compact conformation can also be affected by the surface properties of the irradiated material.

fashion, as shown in Figure 3a. Up to three plateaus were observed in a single plot (within these curves, an average of 65%, 26%, and 9% displayed one, two, and three steps, respectively) regardless of the material surface upon which the HFN molecules were adsorbed. Similar curves, but exhibiting a unique plateau, have previously been reported for molecular motors, such as myosin, and double-helical DNA.47,48 Although for DNA the occurrence of this plateau is still a subject of debate, for myosin it has been attributed to the uncoiling and unfolding of its double α-helical coiled-coil tail. In particular, the coiled-coil myosin was found to extend to about 2.5 times its original length at forces of 20−25 pN. Theoretical predictions and molecular models based on simplified systems suggest that the unfolding pathway of protein domains depends critically on their topology.49,50 Whereas the unfolding pathway of α-helical domains requires only one intrahelix hydrogen bond to be broken at the time, which leads to very low average forces and no observable force peaks, the predicted unfolding pathway for β-sheets involves the simultaneous breakage of a set of interstrand hydrogen bonds, resulting in relatively high average forces. In this latter case, after the hydrogen bonds are concertedly broken, the force is found to rapidly drop due to the increased end-to-end distance of the unfolded domain. Experimentally, this rapid buildup and drop in the force yields the typically reported sawtooh force profiles.29,51,52 The difference between the unfolding behavior of α-helical and β-stranded domains is attributed to the different orientation of the hydrogen bonds relative to the externally applied force vector. In the first case, the hydrogen bonds are colinear to the force vector, whereas in β-stranded domains the hydrogen bonds are perpendicular to it. Therefore, considering that each and every domain that comprises the HFN molecules is only composed of β-sheets, it is highly unlikely that the recorded force plateaus would correspond to the mechanical unfolding of individual domains. It is proposed that the force extension behavior observed in Figure 3a originates from the straightening of an initially compact molecule without the unfolding of its domains. This hypothesis is based on the observation that the maximum distance at which the last recorded plateau ends was consistent with the total length of a single molecule, that is, always below 160−180 nm (Figure 3b). Interestingly, studies of proteolytic and recombinant protein fragments have indicated that the compact conformation of HFN in physiological conditions is stabilized through electrostatic contacts between the FnIII 2−3 domains of one monomer and the FnIII 12−14 domains of the

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CONCLUSIONS In this study, the structure and mechanical stability of HFN have been evaluated in physiological conditions upon adsorption on the nonirradiated and irradiated Ti6Al4V material through the use of atomic force microscopy. The results indicated that the material surface changes occurring E

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(9) Lofidh, H.; Berk, A.; Zipursky, S. L.; Matsudaira, P.; Baltimore, D.; Darnell, J. Molecular Cell Biology, 4th ed.; W. H. Freeman: New York, 2000. (10) Bergkvist, M.; Carlsson, J.; Oscarsson, S. Surface-Dependent Conformations of Human Plasma Fibronectin Adsorbed to Silica, Mica, and Hydrophobic Surfaces, Studied with Use of Atomic Force Microscopy. J. Biomed. Mater. Res., Part A 2003, 64, 349−356. (11) Dickinson, C. D.; Veerapandian, B.; Dai, X.-P.; Hamlin, R. C.; Xuong, N. H.; Ruoslahti, E.; Ely, K. R. Crystal Structure of the Tenth Type III Cell Adhesion Module of Human Fibronectin. J. Mol. Biol. 1994, 236, 1079−1092. (12) Williams, M. J.; Phan, I.; Harvey, T. S.; Rostagno, A.; Gold, L. I.; Campbell, I. D. Solution Structure of a Pair of Fibronectin Type 1 Modules with Fibrin Binding Activity. J. Mol. Biol. 1994, 235, 1302− 1311. (13) Sticht, H.; Pickford, A. F.; Potts, J. R.; Campbell, I. D. Solution Structure of the Glycosylated Second Type 2 Module of Fibronectin. J. Mol. Biol. 1998, 276, 177−187. (14) Potts, J. R.; Campbell, I. D. Fibronectin Structure and Assembly. Curr. Opin. Cell Biol. 1994, 6, 648−655. (15) Johnson, K. J.; Sage, H.; Briscoe, G.; Erickson, H. P. The Compact Conformation of Fibronectin is Determined by Intramolecular Ionic Interactions. J. Biol. Chem. 1999, 274, 15473−15479. (16) Vogel, V. Mechanotransduction Involving Multimodular Proteins: Converting Force into Biochemical Signals. Annu. Rev. Biophys. Biomol. Struct. 2006, 35, 459−488. (17) Craig, D.; Krammer, A.; Schulten, K.; Vogel, V. Comparison of the Early Stages of Forced Unfolding for Fibronectin Type III Modules. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5590−5595. (18) Tskhovrebova, L.; Trinick, J.; Sleep, J. A.; Simmons, R. M. Elasticity and Unfolding of Single Molecules of the Giant Muscle Protein Titin. Nature 1997, 387, 308−312. (19) Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J. M.; Gaub, H. E. Reversible Unfolding of Individual Titin Immunoglobulin Domains by AFM. Science 1997, 276, 1109−1112. (20) Oberhauser, A. F.; Marszalek, P. E.; Erickson, H. P.; Ferndandez, J. M. The Molecular Elasticity of the Extracellular Matrix Protein Tenascin. Nature 1998, 393, 181185. (21) Rief, M.; Pascual, J.; Saraste, M.; Gaub, H. E. Single Molecule Force Spectroscopy of Spectrin Repeats: Low Unfolding Forces in Helix Bundles. J. Mol. Biol. 1998, 286, 553−561. (22) Oberhauser, A. F.; Badilla-Fernandez, C.; Carrion-Vazquez, M.; Fernandez, J. M. The Mechanical Hierarchies of Fibronectin Observed with Single Molecule AFM. J. Mol. Biol. 2002, 319, 433−447. (23) Schlierf, M.; Li, H.; Fernandez, J. M. The Unfolding Kinetics of Ubiquitin Captured with Single-Molecule Force-Clamp Techniques. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7299−7304. (24) Brown, A. E.; Litvinov, R. I.; Discher, D. E.; Weisel, J. W. Forced Unfolding of Coiled-Coils in Fibrinogen by Single-Molecule AFM. Biophys. J. 2007, 92, L39−L41. (25) Carrion-Vazquez, M.; Oberhauser, A. F.; Fowler, S. B.; Marszalek, P. E.; Broedel, S. E.; Clarke, J.; Fernandez, J. M. Mechanical and Chemical Unfolding of a Single Protein: A Comparison. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3694−3699. (26) Best, R. B.; Li, B.; Steward, A.; Daggett, V.; Clarke, J. Can NonMechanical Proteins Withstand Force? Stretching Barnase by Atomic Force Microscopy and Molecular Dynamics Simulation. Biophys. J. 2001, 81, 2344−2356. (27) Brockwell, D. J.; Beddard, G. S.; Clarkson, J.; Zinober, R. C.; Blake, A. W.; Trinick, J.; Olmsted, P. D.; Smith, D. A.; Radford, S. E. The Effect of Core Destabilisation on the Mechanical Resistance of I27. Biophys. J. 2002, 83, 458−472. (28) Yang, G.; Cecconi, C.; Baase, W. A.; Vetter, I. R.; Breyer, W. A.; Haack, J. A.; Matthews, B. W.; Dahlquist, F. W.; Bustamante, C. SolidState Synthesis and Mechanical Unfolding of Polymers of T4 Lysozyme. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 139−144. (29) Smith, D. A.; Brockwell, D. J.; Zinober, R. C.; Blake, A.; Beddard, G. S.; Olmsted, P. D.; Radford, S. E. Unfolding Dynamics of

after the irradiation process largely interfere with the inter- and intramolecular interactions holding together the individual protein domains and stabilizing the compact conformation of the adsorbed HFN molecules. Specifically, it is shown that the molecules becomes structurally looser and mechanically more flexible upon adsorption on the irradiated material. Moreover, force displacement profiles that correlates with the unraveling of a single compact protein without the unfolding of its domains were recorded for the first time. Regardless of the material surface employed for protein adsorption, these profiles displayed force transitions occurring in a stepwise fashion with up to three plateaus observed in a single plot. The recorded transitions have been related with the reported structural details of the compact conformation of HFN in physiological conditions. In addition, the forces involve in the unraveling process have been shown to be generally lower than the forces required to unfold the individual protein domains. All together, this report contributes to our understanding of the HFN remarkable mechanical properties and highlights the potential of using physico-chemically regulated surface properties to tune the mechanical stability of proteins.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: (+34) 924 289300 ext. 86173. Fax: (+34) 924 289651. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge financial support from the “Ministerio de Ciencia e Innovación” (MAT2009-14695-C0401) and “Junta of Extremadura-FEDER: European Regional Development Fund” (GR10149). V.V.-R. thanks the “Ministerio de Ciencia e Innovación” for the Ramón y Cajal fellowship (RYC-2008-03482).

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REFERENCES

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dx.doi.org/10.1021/la401776e | Langmuir XXXX, XXX, XXX−XXX