Molecular Orientation of Tropoelastin is Determined by Surface

Article pubs.acs.org/Biomac

Molecular Orientation of Tropoelastin is Determined by Surface Hydrophobicity Anton P. Le Brun,† John Chow,‡ Daniel V. Bax,‡ Andrew Nelson,† Anthony S. Weiss,‡ and Michael James*,†,§ †

Bragg Institute, Australian Nuclear Science and Technology Organisation, Locked Bag 2001, Kirrawee DC, NSW 2232, Australia School of Molecular Bioscience, University of Sydney, Sydney, NSW 2006, Australia § School of Chemistry, University of New South Wales, Kensington, NSW 2052, Australia ‡

S Supporting Information *

ABSTRACT: Tropoelastin is the precursor of the extracellular protein elastin and is utilized in tissue engineering and implant technology by adapting the interface presented by surface-bound tropoelastin. The preferred orientation of the surface bound protein is relevant to biointerface interactions, as the C-terminus of tropoelastin is known to be a binding target for cells. Using recombinant human tropoelastin we monitored the binding of tropoelastin on hydrophilic silica and on silica made hydrophobic by depositing a self-assembled monolayer of octadecyl trichlorosilane. The layered organization of deposited tropoelastin was probed using neutron and X-ray reflectometry under aqueous and dried conditions. In a wet environment, tropoelastin retained a solution-like structure when adsorbed on silica but adopted a brush-like structure when on hydrophobized silica. The orientation of the surface-bound tropoelastin was investigated using cell binding assays and it was found that the C-terminus of tropoelastin faced the bulk solvent when bound to the hydrophobic surface, but a mixture of orientations was adopted when tropoelastin was bound to the hydrophilic surface. Drying the tropoelastin-coated surfaces irreversibly altered these protein structures for both hydrophilic and hydrophobic surfaces.

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Due to the inherent insolubility and flexibility of elastin,11−13 structural analysis has focused on studies of tropoelastin in solution;13,14 while electron microscopy of deposited tropoelastin has given only limited information on the shape of the protein.15 Small-angle X-ray scattering (SAXS) corroborated with small angle neutron scattering (SANS) of tropoelastin and its subfragments revealed that tropoelastin adopts a defined nanostructure that consists of specific modules that specialize in elasticity and dermal fibroblast interactions.16 Atomic force microscopy and photoelectron spectroscopy studies of elastinlike peptides immobilized to silica showed that the structure of adsorbed chains differs from that in solution, but as they correspond to small parts of the protein, their relevance to tropoelastin is doubtful.17,18 Surface-bound tropoelastin is being tested in tissue engineering and implant technology19 bound to hydrophobic surfaces such as polyethylene glycol terephthalate (PTFE),20 hydrophilic surfaces such as oxidized polystyrene21 and graded metal surfaces.22,23 Three-dimensional scaffolds made by electrospinning and in the form of hydrogels24−26 support cell growth, while tropoelastin has been covalently bonded to plasma polymer coated stainless steel surfaces for use in coronary stents.27,28 Elastin-coated surfaces have been shown to affect

INTRODUCTION Elastin is the extracellular matrix protein primarily responsible for imparting elasticity to tissues that experience extension and contraction. In humans, elastin forms over 90% of the elastic fibers found in tissues such as arteries, lung and the skin dermis.1 Elastin is stable in adult humans with a very low turnover rate,2,3 and while its regeneration can be stimulated in adult organisms, they are generally ill-equipped to do so.4,5 Synthetic elastin is therefore an important biomaterial for tissue engineering and repair.6 Elastin is assembled from its soluble precursor, tropoelastin, a 60 kDa monomer protein that assembles into elastin fibers by a reversible self-aggregation process called coacervation.6 Tropoelastin is composed of alternating hydrophilic and hydrophobic domains. Hydrophilic domains are characterized by their high lysine and alanine contents and play roles in crosslinking processes.7 The hydrophobic domains are enriched by nonpolar residues valine, glycine, and proline that typically occur in repeating motifs. While the tertiary structure of tropoelastin has not yet been definitively determined, analysis of individual domains indicates secondary structures such as polyproline II (PPII) and disordered structure.8,9 The C-terminus region of tropoelastin has been shown to play a key role in the assembly into elastin fibers.10 Characteristic features of this region include the only two cysteine residues in the protein, which forms a disulfide bond and its termination in a positively charged RKRK sequence.7 © 2011 American Chemical Society

Received: October 9, 2011 Revised: December 5, 2011 Published: December 17, 2011 379

dx.doi.org/10.1021/bm201404x | Biomacromolecules 2012, 13, 379−386

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Table 1. Parameters for Tropoelastin number of amino acids

molecular mass (g mol−1)

theoretical protein volume (Å3)

theoretical pI

theoretical nSLD in D2O (×10−6 Å−2)

theoretical nSLD in H2O (×10−6 Å−2)

theoretical xrSLD (×10−6 Å−2)

number of exchangeable protons

698

60017

75755

10.4

2.71

1.74

11.9

793

cell activity and proliferation,29,30 suggesting that they may be useful for the treatment of dermal and vascular conditions. Such surfaces are of particular interest in cases where tissue regeneration has proven problematic due to the slow regeneration of elastin in mammalian adults. In this paper, we describe the presentation of human tropoelastin when it is adsorbed onto representative hydrophilic and hydrophobic surfaces. Flat surfaces are required to facilitate analysis by X-ray and neutron reflectometry, so in these studies the hydrophilic surface was silica and the hydrophobic surface was a self-assembled monolayer of octadecyl trichlorosilane (OTS) on a silicon substrate. Tropoelastin adsorption was monitored by quartz crystal microbalance with dissipation (QCM-D), and the internal structure of the tropoelastin layer was studied along the axis perpendicular to the surface (the z-axis) under near-physiological buffer conditions by neutron reflectometry (NR) and in the dried state exposed to air using X-ray reflectometry (XRR) and atomic force microscopy (AFM). The orientation of the surface-adsorbed tropoelastin was investigated using cell binding assays.

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the same conditions. The continuous flow of solutions into the samples cells was controlled by a peristaltic pump (Ismatec SA, Glattbrug, Switzerland). Sensor crystals coated with SiO2 (QSX-303 from Q-sense) were used. The crystals were UV cleaned for 10 min followed by washing with 1% (w/v) SDS and rinsing with Milli-Q water. Immediately prior to use, the crystals were UV cleaned for an additional 10 min. OTS-functionalized sensor crystals were produced from SiO2-coated sensor crystals using the OTS-deposition protocol described below, and their functionally was verified after OTS treatment. Protein was adsorbed on the surfaces of the OTS-coated and unmodified QCM-D sensor crystals. Tropoelastin at a range of different concentrations was allowed to flow over each surface at 50 μL min−1 at 24 °C under continuous flow. After saturation, the surfaces were washed with PBS at 50 μL min−1 to remove nonadsorbed protein. Data were modeled using the Voigt model35 with QTools software. A leastsquares fitting was carried out and the data were fitted with a model incorporating thickness, viscosity, and shear modulus parameters. Surface Preparation. For neutron and X-ray reflectivity experiments, n-type silicon (111) wafers of dimensions 80 × 40 × 15 mm were cleaned for 1 h at 85 °C in a solution of H2O/H2SO4/H2O2 (4:3:1 by volume). Wafers were rinsed in Milli-Q water, dried, and then UV cleaned for 20 min before washing sequentially with water and propan-2-ol, followed by drying under a stream of nitrogen gas. OTS-coated silicon wafers were produced by incubating the wafers in a solution of 1 mM OTS in n-hexadecane within an airtight Teflon reaction vessel at 25 °C overnight. The coated silicon wafers were sequentially washed with dichloromethane, propan-2-ol, and water before drying under a stream of nitrogen gas. Neutron Reflectometry Measurements and Modeling. Silicon wafers coated with tropoelastin were assembled into aluminum sample cells with a Teflon backing plate that incorporated a solvent reservoir and inlet/outlet tubes to allow for solvent/sample exchange. NR data were measured using the Platypus time-of-flight neutron reflectometer and a cold neutron spectrum (2.8 Å ≤ λ ≤ 18.0 Å) at the OPAL 20 MW research reactor (Sydney, Australia).36,37 Neutron pulses (20 Hz) were generated using a disk chopper system (EADS Astrium GmbH, Germany) in the low resolution mode (Δλ/λ = 8%) and recorded on a 2-dimensional helium-3 neutron detector (Denex GmbH, Germany). Reflected beam spectra were collected for each of the surfaces at 0.8° for 1 h (0.6 mm slits) and 3.0° for 3 h (2.25 mm slits), respectively. Direct beam measurements were collected under the same collimation conditions for 1 h each. The data was reduced and scaled using the SLIM module of the Platypus control software.38 Structural parameters for the silica, OTS and protein layers were refined using MOTOFIT39 reflectometry analysis software with reflectivity data as a function of the momentum transfer vector Q (=4π(sin θ)/λ). In the fitting routines, the genetic algorithm was selected to minimize χ2 values by varying the thickness, roughness, and neutron scattering length density (nSLD) of each layer. Values of the neutron scattering length density used in this study are calculated using the following equation:

EXPERIMENTAL SECTION

Materials and Methods. Materials. All chemicals were purchased from Sigma-Aldrich unless otherwise stated. Silicon wafers were purchased from Crystran Ltd. (Poole, Dorset, U.K.). Deuterium oxide (D2O) was purchased from Atomic Energy of Canada Ltd. (Sheridan Park, ON, Canada). Protein Preparation. Recombinant tropoelastin was expressed and purified in-house as previously described.31−33 The lyophilized protein was dissolved in phosphate-buffered saline (PBS, 10 mM sodium phosphate pH 7.0, 150 mM NaCl, 0.22 μm filtered and degassed). To investigate the effects of salt on surface binding the sodium chloride concentration was varied from 0 to 500 mM. For investigating pH effects, 10 mM sodium phosphate was used for pH 7.0−8.0, and 10 mM glycine was used for all other pH values, with the NaCl concentration remaining 150 mM. Insoluble aggregates were removed by centrifugation at 14000 rcf for 15 min. The properties of tropoelastin are summarized in Table 1. We inputted the primary sequence of tropoelastin into the ProtParam tool of www.expasy.org34 to calculate the molecular mass. The number of exchangeable protons was derived by adding the exchangeable protons for each amino acid residue. The theoretical pI for tropoelastin was calculated using the ExPASY routine. The protein volume was determined by adding the volumes of the amino acid residues. Multiangle Laser Light Scattering. Multiangle laser light scattering (MALLS) data were obtained for purified tropoelastin dialyzed in PBS. The MALLS system comprised of a Superdex 200 10/30 gel filtration column driven by an Ä KTA FPLC platform, feeding into a Wyatt Technology miniDAWN light scattering unit and an Optilab DSP refractometer. The system was pre-equilibrated with 5 column volumes of buffer. To determine the relative molecular mass (Mr) of tropoelastin, the system was calibrated to an absolute scale using the intrinsic Rayleigh scattering of toluene. A uniform refractive index to concentration gradient (dn/dc) of 0.19 mL g−1 was assumed, and the calibration was verified using a bovine serum albumin standard. Quartz Crystal Microbalance with Dissipation (QCM-D). QCM-D measurements were carried out on a Q-sense E4 instrument (Q-sense, Gothenburg, Sweden). This instrument possesses four sample cells that enables four experiments to be performed simultaneously under

nSLD = NA ∑ (p /A i)bi

(1)

where NA is Avogadro’s number, p is the mass density, Ai is the atomic mass, and bi is the coherent neutron scattering length40 of component i. The number of exchangeable protons (736, Table 1) were also used in this calculation. When in D2O, we assumed that all of the exchangeable protons were replaced by D atoms, and a value of b = 6.671 fm was used for these atoms in the calculation of nSLD. When in H2O, a value of b = −3.739 fm was used for H in its naturally abundant form. X-ray Reflectometry. Following neutron reflectometry measurements, XRR profiles were measured under ambient conditions from 380


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dried samples using a Panalytical Ltd X′Pert Pro reflectometer (Cu Kα radiation: λ = 1.54056 Å). The X-ray beam was focused using a Göbel mirror and collimated with a 0.1 mm presample slit and postsample parallel collimator. Reflectivity data were collected over the angular range 0.05° ≤ θ ≤ 5.00°, with a step size of 0.010° and counting times of 10 s per step. Observed data are shown as points, and calculated reflectivity based on refined structural models are shown as solid lines. Structural parameters were refined using MOTOFIT analysis software. In this instance, the X-ray scattering length density xSLD was calculated using the equation:

xSLD = NAre ∑ (p /A i)Zi

Adsorption of Tropoelastin to Hydrophilic and Hydrophobic Silicon Surfaces. Initial measurements used a quartz crystal microbalance with dissipation (QCM-D).43 The general procedure followed that tropoelastin was incubated on the surface until saturation for the particular conditions was reached. Figure 1a shows the change in frequency (Δf) of the

(2)

where NA is Avogadro’s number, re is the Bohr electron radius (2.818 × 10−15 m), p is the mass density, Ai is the atomic mass, and Zi is the atomic number of component i. Cell Attachment Analysis. The cell attachment methodology was as outlined in ref 41. The 0.8 × 1.2 cm samples were coated with 20 μg mL−1 tropoelastin for 16 h at 4 °C. Where stated, nonspecific silicon binding was blocked with 10 mg mL−1 heat denatured BSA (80 °C for 10 min, then cooled on ice) in PBS for 1 h at room temperature. Near confluent 75 cm2 flasks of human dermal fibroblasts were harvested by trypsinisation, and the cell density adjusted to 2 × 105 cells mL−1. The BSA blocking solution was aspirated, the wells washed with PBS and 1 mL aliquots of the cell suspension were added for 60 min and allowed to adhere to the surface at 37 °C in a 5% CO2 incubator. The nonadherent cells were then removed. To estimate percentage cell attachment a minimum of three known cell number controls were plated by adding 0, 0.5, or 1 mL of cells to unblocked plasma immersion ion implantation (PIII) treated polystyrene samples which possess high cell binding affinity. After incubation, the cells in the cell number controls were fixed with the addition of 100 μL of 50% glutaraldehyde (w/v) directly to the media. The media was removed from the experimental wells and nonadherent cells were removed with two 0.75 mL PBS washes. The samples were fixed with the addition of 0.75 mL of 5% glutaraldehyde (w/v) in PBS for 20 min, then washed with three 1 mL aliquots of PBS. Bound cells were stained with 500 μL of 0.1% (w/v) crystal violet in 0.2 M MES pH 5.0 for 1 h at room temperature before washing extensively with dH2O. A total of 500 μL of 10% (v/v) acetic acid was added and the absorbance measured at 570 nm using a Bio-Rad MPLATE model M680. The percentage cell standards were plotted and a linear regression fitted which was used to convert experimental absorbances into percentage attachment. In all experiments, triplicate measurements were taken. Atomic Force Microscopy (AFM). AFM measurements were acquired in tapping−mode in air using a Nanoscope Multimode atomic force microscope (Digital Instruments) and commercially modified Si3N4 cantilevers. Several images were taken such that the parameters of force, tip frequency, scan size, and scanning speed could be optimally adjusted for best imaging. All measurements were performed at room temperature and the laser used for detecting the cantilever deflection had a minimal effect on the temperature of the sample. The height distribution was determined by crosssection analysis using the default Digital Instruments (Version 4.31, Rev B.) software.

Figure 1. (a) QCM-D traces showing the changes in frequency on the fifth overtone following tropoelastin adsorption onto unmodified silica (circles) and an OTS (squares) surface. (b) The mass of tropoelastin adsorbed to silica (circles) and OTS (squares) surfaces as a function bulk protein concentration. Tropoelastin was in 10 mM sodium phosphate pH 7.0 and 150 mM NaCl buffer. The error bars are the standard error of the mean and the solid curves are based on exponential fits to these data and are intended only to indicate trends.

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RESULTS Tropoelastin is a Monomer in the Preadsorption Solution. At physiological temperatures, tropoelastin undergoes a reversible self-aggregation process called coacervation.6,42 To ensure that the tropoelastin used in this study was present as a monomer in solution, MALLS was used to estimate the mass, oligomerization and coacervation state of the protein (Supporting Information, Figure S1). The measured molecular mass of the tropoelastin from these data was 56240 g mol−1, which is within experimental error of the expected 60 kDa expected for this protein. This result confirms that the tropoelastin was a monomer under the solution and temperature conditions used in this study.

fifth overtone as tropoelastin (100 μg mL−1) is adsorbed onto the hydrophilic silica surface (circles) and onto the hydrophobic OTS-coated wafer (squares). A total incubation time of 25 min was typically sufficient for complete saturation. After incubation, the tropoelastin-bound surfaces were washed with PBS and both QCM-D traces show that the rinsing process removes some tropoelastin from the surfaces (Figure 1a). The amount of tropoelastin lost from the surface upon rinsing was significantly less than compared to the amount of protein that remained adsorbed. The overtones were spread out (Supporting Information, Figure S2) showing that surface adsorbed 381


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tropoelastin is a nonrigid structure with associated water and therefore the Sauerbrey equation cannot be used straightforwardly to calculate protein parameters. When the complete QCM-D was used, the Voigt-model35 of a viscoelastic solid was used to calculated the masses on the surface. Figure 1a clearly indicates that after rinsing with PBS substantially more tropoelastin is bound to the silica surface (Δf = −43 Hz, 7.7 mg m−2) than to the OTS-functionalized hydrophobic surface (Δf = −27 Hz, 4.6 mg m−2). When the complete set of QCM-D data was used (Supporting Information, Figure S2), the Voigt-model was used to characterize the protein thickness on the surface after the PBS wash, giving layer thicknesses of 230 and 220 Å on the silica and OTS-coated surfaces, respectively. Different protein concentrations were also used to investigate tropoelastin adsorption onto the hydrophilic silica surface and the hydrophobic OTS monolayer. The protein was in a PBS buffer consisting of 150 mM NaCl at pH 7.0, resulting in tropoelastin having a net positive charge due to a pI of 10.4. This enabled the tropoelastin to adsorb to the negatively charged surface of untreated silica, while the hydrophobicity of the protein enabled it to adsorb to the OTS-coated surface. The concentration range was from 1 to 100 μg mL−1, and the average adsorbed amount onto four equivalent surfaces is shown in Figure 1b. At the higher concentrations, large frequency shifts were observed during the incubation of tropoelastin to both types of surface that indicated successful adsorption of tropoelastin (Supporting Information, Figure S3). As the bulk tropoelastin concentration decreased the frequency shifts in the QCM-D data also reduced indicating less binding. Both types of surfaces showed saturation of tropoelastin coverage at 25 μg mL−1. In all instances, the silica surface had more tropoelastin adsorbed to it than to the OTS surface (Figure 1b). The effect of salt content and pH of the bulk solutions on tropoelastin binding was also investigated (Figure 2 and Supporting Information, Figures S4 and S5). Tropoelastin at a concentration of 20 μg mL−1 was used throughout these experiments. At pH 7, little change was observed in tropoelastin adsorption onto the silica surface for NaCl concentrations below 300 mM, with the mass bound being ∼6.0 mg m−2 (circles, Figure 2a). At NaCl concentrations above 350 mM, a dramatic decrease in the mass of tropoelastin bound to the silica surface was seen showing that the adsorption was charge dependent. Tropoelastin was found to adsorb to the hydrophobic OTS surface at all NaCl concentrations used (squares, Figure 2a). Thus, tropoelastin adsorbed to OTS via hydrophobic regions of the protein and was not dependent on electrostatics. There was a slight increase in the mass of tropoelastin adsorbed to the OTS with increasing NaCl concentration. By way of comparison, Holst et al.21 recently examined the adsorption of tropoelastin onto oxidized polystyrene using QCM-D. At an equivalent concentration (20 μg/mL) in PBS, these authors also found an adsorbed monolayer of tropoelastin, with an adsorbed mass of ∼430 ng/cm2. This value is between that of for our hydrophilic silica surface (∼600 ng cm−2) and the hydrophobic OTS surface (∼350 ng cm−2). Varying the pH of the buffer used from pH 7.0 to 9.0, while keeping the NaCl concentration constant at 150 mM, had essentially no effect on tropoelastin adsorption onto both the silica (circles) and OTS (squares) surfaces (Figure 2b). These pH values were less than the theoretical pI of tropoelastin and, therefore, are optimal for tropoelastin adsorption to silica

Figure 2. Absorption of tropoelastin to silica (circles) and OTS (squares) surfaces as a function of (a) bulk NaCl concentration and (b) bulk pH. The tropoelastin concentration used was 20 μg mL−1. For the NaCl experiments, 10 mM sodium phosphate at pH 7.0 was used. For the pH experiments, the NaCl concentration was 150 mM. The error bars are the standard error of the mean. Solid curves in (a) are based on a sigmoidal fit (for silica) and a linear fit (for OTS) and are intended only to indicate trends.

(Figure 2b). A moderate increase in tropoelastin adsorption to both types of surfaces was observed at pH 10.0 (Figure 2b). This pH was very close to the theoretical pI of tropoelastin (10.4) and the overall charge of the protein would have been close to neutral. This meant that there was less electrostatic repulsion between tropoelastin molecules allowing for closer packing and hence a higher absorption. At pH 11.0, tropoelastin has a slight overall negative charge which was not favorable for adsorption to silica, as seen by the substantial decrease in mass adsorption to ∼0.5 mg m−2 (Figure 2b). Tropoelastin still bound to the OTS surface at pH 11.0 showing that the adsorption to the hydrophobic surface was not dependent on electrostatics between the surface and the protein. In bulk solutions, self-association of tropoelastin molecules due to hydrophobic interactions has also been observed at pH 11, where charge repulsions are minimized by removing charge from the lysine residues.44 Structure of Surface-Bound Tropoelastin in PBS. Surface-bound tropoelastin immersed in PBS was analyzed at the solid−liquid interface using neutron reflectometry, a technique which enables the structure of surface-bound proteins to 382


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bound surface (silica or OTS) and a tropoelastin-coated surfaces (Figure 3), confirming that tropoelastin bound to both hydrophilic or hydrophobic surfaces as indicated by the QCMD data. To generate a structural model of each surface, the reflectivity data were fitted using a genetic algorithm in which constraints can be imposed on each parameter that is to be modeled. Although a single-layer model fitted the reflectivity profile of adsorbed tropoelastin (χ2 = 0.0604) giving calculated area per molecule for tropoelastin as 1504 and 1924 Å2 for silica-bound and OTS-bound surfaces, the use of a multilayer model resulted in significantly better fits to the data (χ2 = 0.00195), suggesting that surface-bound tropoelastin did not have an internally homogeneous structure. Tropoelastin adsorbed to silica (152 ± 7 Å) formed a surface similar in thickness to tropoelastin adsorbed to OTS (158 ± 8 Å), with both thicknesses less than the ∼200 Å maximum dimension of tropoelastin in solution.16 Different models were needed for each surface to describe the structure of surface-bound tropoelastin. As evidenced by the lower χ2 values, a three-layer model best described silica-bound tropoelastin showing a dense protein layer close to the substrate surface, followed by a more solvated layer finally with a less solvated layer adjacent to the bulk solvent (Table 2). In contrast, tropoelastin adsorbed to the OTS surface was best described using a two layer model with a thin layer (11 ± 3 Å) of dense protein at the protein−OTS interface followed by a layer of less densely packed protein toward bulk solvent. The increasing solvation of the layers depended on their distance from the OTS, as expected for a model where OTS-bound tropoelastin had a brush structure that radiated from the OTS surface. We have previously shown that the ability of tropoelastin to support fibroblast adhesion is modulated by the hydrophobicity of the underlying polymer.41 While NR can provide an overall structural model for tropoelastin adsorbed on the different surfaces there, is no specific structural evidence for the orientation of the tropoelastin N- and C-termini. We have used a cell attachment assay (based upon the technique of Bax et al.41) for the different surfaces used in this study so that the orientation of tropoelastin N- and C-termini can be inferred. The Cterminus of tropoelastin is a hydrophilic αvβ3 integrin binding domain. The exposure of which, to the bulk solvent, will support fibroblast adhesion to this tropoelastin surface.46 To test the orientation of tropoelastin, we determined the cellbinding properties of tropoelastin coated onto untreated and OTS-treated silicon (Figure 4). In the absence of tropoelastin protein coating, silica and OTS-treated silicon supported 69 ± 11 and 36 ± 1% dermal fibroblast adhesion, respectively. Bovine serum albumin (BSA) blocked dermal fibroblast adhesion to 19 ± 1 and 8 ± 1% for silica and OTS-treated silicon. This BSA blocking allowed measurement of cell adhesion to bound proteins without interference due to cell binding to the underlying silica or OTS substrates. On the tropoelastin-coated

be studied while submerged in solvent. The use of neutron reflectometry also allows the difference in neutron scattering lengths between hydrogen (b = −3.741 fm) and its isotope deuterium (b = +6.671 fm) to be exploited to improve the contrast between layers in soft matter systems. This is done by measuring the neutron reflectivity of an adsorbed protein (abundant in hydrogen) in a buffer solution made up in deuterium oxide (D2O). As tropoelastin is rich in alanine, glycine, and proline residues, it has a comparatively low neutron scattering length density (nSLD) compared to many proteins (Table 1) and, hence, presents a good contrast against the surrounding D2O solvent subphase. Neutron reflectometry experiments were conducted on tropoelastin adsorbed to two different surfaces: the electronegative and hydrophilic oxide surface of the untreated silicon substrate; and the hydrophobic surface of a silicon wafer coated with a 24 ± 1 Å thick uniform layer of OTS with a high surface coverage (>95%) across the oxide surface (Figure 3b and

Figure 3. Reflectivity profiles for tropoelastin adsorbed onto (a) a silica and (b) an OTS surface. Blue points with error bars are the bare surfaces measured in D2O. Red points represent the adsorbed tropoelastin measured in D2O where the curves represent the fit to the multilayer models.

Supporting Information, Figure S6) consistent with published results for an OTS monolayer.45 Tropoelastin dissolved in PBS adsorbed to these surfaces during incubation for 1 h at 24 °C, after which the wafers were then gently rinsed with PBS to remove any nonadsorbed protein. Reflectivity profiles were collected in PBS made from D2O, and showed substantial differences between the nonprotein

Table 2. Fit Parameters for Tropoelastin Derived from the Neutron Reflectivity Dataa silica/tropoelastin/D2O

a

OTS/tropoelastin/D2O

parameter

layer 1

layer 2

layer 3

total

layer 1

layer 2

total

thickness (Å) nSLD in D2O (×10−6 Å−2) protein volume fraction

49 ± 3 4.6 ± 0.1 0.47

33 ± 3 5.5 ± 0.1 0.24

70 ± 4 4.7 ± 0.2 0.43

152 ± 7

11 ± 3 4.6 ± 0.2 0.47

147 ± 5 5.3 ± 0.2 0.29

158 ± 8

Errors represent ± one standard deviation as reported from the fitting in MOTOFIT. 383


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dense layer regardless of whether it was bound to a hydrophilic or hydrophobic surface. Such results are in excellent agreement with AFM measurements of the dry tropoelastin surface on the OTS monolayer (Supporting Information, Figure S7).

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DISCUSSION Tropoelastin is a monomer in solution under these buffer conditions where it is an asymmetric molecule.16 The protein binds to hydrophilic and hydrophobic surfaces as observed for other proteins that bind to silica and OTS surfaces;47−54 however, its orientation and shape following binding cannot be readily predicted from its solution structure. Tropoelastin adsorbed as a monolayer on both silica and OTS surfaces which is consistent with other QCM-D measurements observed for tropoelastin binding to different surfaces such as oxidized polystyrene21 and shows that tropoelastin readily forms monolayers on surfaces. The monolayers were measured through treatment of the QCM-D and neutron reflectivity data. In an aqueous environment, the total bound tropoelastin layer thickness was 150−160 Å, which was approximately 70−75% of the maximum tropoelastin length in solution, as the SAXS structure calculates Dmax (the maximum dimension) to be equal to 210 Å.16 Thus, the more compact surface structure of tropoelastin was consistent with monolayer formation. In support, the thickness of the tropoelastin modeled from the QCM-D data was close to the Dmax of the solution structure. However, this was a maximum estimate of thickness due to the dissipation of each of the overtones (Supporting Information, Figure S2). When the protein coated surfaces dried out in air, a dense protein layer around 60 Å thick was found regardless of the surface properties (Figure 5). This is the same thickness of a tropoelastin monolayer covalently bonded to a plasma coated stainless steel surface in air,27 confirming that tropoelastin consistently formed a monolayer on surfaces regardless of the surface properties. Under aqueous conditions, the adsorbed tropoelastin monolayer presented a heterogeneous structure along the axis perpendicular to the surface (Table 2 and Figure 3). When adsorbed onto hydrophilic silica, tropoelastin presented a less extended structure than in solution and formed a dense protein layer close to the surface. These findings correlated with results from other proteins, for example, the globular protein lysozyme unfolds when adsorbed to an OTS monolayer, where it presents a dense layer close to the surface topped by a substantial less dense layer at the bulk solution interface53 but retains its solution structure when it is adsorbed to silica.47 In contrast, β-casein that is adsorbed to OTS surface forms a brush-like structure48,54 that is similar to that seen here for tropoelastin. However, unlike tropoelastin, β-casein forms an asymmetric bilayer structure when bound to silica.49 The tropoelastin neutron reflectivity data are best explained by a model where, on silica, tropoelastin preferentially binds through either end of the molecule, while on OTS, tropoelastin binds along the length of the molecule with one end folded outward (Figure 6). The C-terminal domain of tropoelastin has a hydrophilic αvβ3 integrin-binding motif,46 which can bind dermal fibroblast cells. Fibroblast adhesion assays were conducted to test for the orientation of the tropoelastin on different surfaces. As the C-terminus is hydrophilic, the tropoelastin orientates on the OTS surface such that the C-terminal faces toward the bulk solvent. This orientation was confirmed by the higher fibroblast attachment seen on the OTS surface (Figure 4). There is a possibility that a mixture of structures could be formed on the

Figure 4. Human dermal fibroblast attachment to untreated (gray bars) or OTS-treated (black bars) silicon. Samples were uncoated (left), BSA blocked (center), or tropoelastin-coated and BSA-blocked (right bars). Error bars indicate standard deviations of triplicate measurements.

OTS surface (after BSA blocking), dermal fibroblast adhesion showed 75 ± 10% adhesion; substantially higher than for the OTS-treated silicon (36%) or the BSA blocked OTS surface (8%). In contrast, tropoelastin bound to untreated silica supported only 26 ± 7% dermal fibroblast adhesion, which was within errors of the adhesion to the BSA block control (19%). Adsorbed Tropoelastin Has a Collapsed Structure in Air. The structure of tropoelastin adsorbed onto surfaces in air was measured using X-ray reflectometry (Figure 5). As with the

Figure 5. X-ray reflectivity of tropoelastin adsorbed onto silica (blue points) and OTS (red points) in air. The lines are a multilayer model fit to the data.

neutron data, multilayer models were used to fit the XRR data though these layers were substantially thinner than those of tropoelastin in PBS buffer solutions. The total thickness for tropoelastin adsorbed onto OTS and silica was 60 ± 3 and 44 ± 3 Å, respectively. Compared with surface-bound tropoelastin in solvent, the structure of silicon-bound tropoelastin in air was that of a collapsed chain with a volume fraction of 0.9. While tropoelastin adsorbed to OTS still retained a thin layer of protein at the protein-OTS interface the volume fraction of the protein was 0.7. These results would be expected if the structure of dried tropoelastin collapsed into a 384


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ASSOCIATED CONTENT

S Supporting Information *

Supporting Figures S1−S5. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +612 9717 9299. E-mail: [email protected]. au.

Figure 6. Models of tropoelastin on a hydrophilic silica surface showing that tropoelastin retains its solution structure and tropoelastin adopting a brush structure on a hydrophobic OTS surface with the C-terminal domain exposed to the bulk solution.

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ACKNOWLEDGMENTS The authors thank Marie Gillon for technical assistance. A.P.L.B. is funded by the ANSTO executive. A.S.W. acknowledges grant support from AINSE, Australian Research Council, the National Health and Medical Research Council, and the Defence Health Foundation.

OTS surface, where there are some tropoelastin molecules lying flat on the surface interspersed with tropoelastin molecules having the long axis normal to the surface. This model is unlikely, as tropoelastin lying flat on a hydrophobic surface is not expected to be energetically favorable. If a mixed structure was the case, then a higher tropoelastin volume fraction would be observed in the layer adjacent to the substrate, with a lower volume fraction into the bulk. On the silica surface, the tropoelastin adsorbed in both orientations, with less of the C-terminus being exposed, resulting in lower fibroblast attachment. This result corresponded with fibroblast attachment to tropoelastin-coated PTFE surfaces, which were hydrophobic or treated to make them hydrophilic.41 Figure 6 shows our model for tropoelastin assembly and orientation onto hydrophilic and hydrophobic silicon. Tropoelastin absorption to solid surfaces has value in applications that include implant technologies and tissue engineering.19 Substrate and subphase properties could affect the presentation of the adsorbed protein. We find here that surface hydrophobicity can alter the structure of surface-bound tropoelastin (Table 2) as OTS-bound tropoelastin presented an unfolded brush structure (Figure 3b). Biophysical analysis of tropoelastin covalently bound to plasma-coated stainless steel was conducted in air and showed a thin collapsed monolayer;27 while under aqueous conditions, tropoelastin is bioactive,27,28 indicating that surface-bound tropoelastin readily converts between folding states when transferred from a dry to an aqueous environment. In support of this ability to refold when exposed to an aqueous environment, tropoelastin can be deposited under evaporative conditions during electrospinning, then cope with hydration to promote activity and proliferation.29 Similarly, the elasticity of elastin is absolutely dependent on an aqueous environment, whereas the protein is brittle when dried.

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REFERENCES

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CONCLUSIONS

Tropoelastin monomer in solution was found to bind to both hydrophilic silica and hydrophobic OTS surfaces. The bound tropoelastin adopted different structures that depend on the properties of the surface and hydration state of the system. Tropoelastin on silica formed a monolayer where the protein shape was close to that of the solution structure. However, when adsorbed to OTS, tropoelastin adopted a brush-like structure with a dense protein layer close to the hydrophobic surface and the C-terminus facing the bulk solution. When either protein surface was dried, the structures at the solid−liquid interface were lost and collapsed monolayer were observed. 385


Biomacromolecules

Article

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Molecular Orientation of Tropoelastin is Determined by Surface

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