Article pubs.acs.org/Langmuir

Molecular Structure and Equilibrium Forces of Bovine Submaxillary Mucin Adsorbed at a Solid−Liquid Interface Bruno Zappone,*,† Navinkumar J. Patil,§ Jan B. Madsen,‡ Kirsi I. Pakkanen,‡ and Seunghwan Lee*,‡ †

Consiglio Nazionale delle Ricerche - Istituto di Nanotecnologia (CNR-Nanotec) and LICRYL c/o Dipartimento di Fisica, Università della Calabria, cubo 33/B, Rende (CS) 87036 Italy § Dipartimento di Fisica, Università della Calabria, cubo 31/C, Rende (CS) 87036 Italy ‡ Department of Mechanical Engineering, Technical University of Denmark, DK-2800, Kgs. Lyngby, Denmark ABSTRACT: By combining dynamic light scattering, circular dichroism spectroscopy, atomic force microscopy, and surface force apparatus, the conformation of bovine submaxillary mucin in dilute solution and nanomechanical properties of mucin layers adsorbed on mica have been investigated. The samples were prepared by additional chromatographic purification of commercially available products. The mucin molecule was found to have a z-average hydrodynamic diameter of ca. 35 nm in phosphate buffered solution, without any particular secondary or tertiary structure. The contour length of the mucin is larger than, yet of the same order of magnitude as the diameter, indicating that the molecule can be modeled as a relatively rigid polymeric chain due to the large persistence length of the central glycosylated domain. Mucin molecules adsorbed abundantly onto mica from saline buffer, generating polymer-like, long-ranged, repulsive, and nonhysteretic forces upon compression of the adsorbed layers. Detailed analysis of such forces suggests that adsorbed mucins had an elongated conformation favored by the stiffness of the central domain. Acidification of aqueous media was chosen as means to reduce mucin−mucin and mucin−substrate electrostatic interactions. The hydrodynamic diameter in solution did not significantly change when the pH was lowered, showing that the large persistence length of the mucin molecule is due to steric hindrance between sugar chains, rather than electrostatic interactions. Remarkably, the force generated by an adsorbed layer with a fixed surface coverage also remained unaltered upon acidification. This observation can be linked to the surface-protective, pH-resistant role of bovine submaxillary mucin in the variable environmental conditions of the oral cavity. properties for the hydration,7 mucoadhesion,10 and lubrication8,11,12 of various engineering materials. The aim of the present study is to investigate the relationship between structural features of single mucin molecules and nanoscale mechanisms of surface force generation by adsorbed mucin layers, which underlie surface-protecting and lubricating properties. In order to minimize the influence of nonmucin impurities in commercial BSM samples, known to deeply affect surface properties,13 additional anion exchange chromatographic purification was conducted.14 Dynamic light scattering (DLS), circular dichroism (CD) spectroscopy, and atomic force microscopy (AFM) were used to characterize structural features of isolated BSM molecules, namely, the hydrodynamic radius, conformation, and contour length in dilute solutions, respectively. Normal force measurements were conducted using the surface force apparatus (SFA) on BSM layers adsorbed on mica from concentrated solution at physiological salinity. Previous SFA studies on mucins have focused on rat

1. INTRODUCTION Mucins are a major component of the mucus gel that coats the epithelial surface of many biological organs and tissues, including mouth, eyes, articular joints, and gastrointestinal, respiratory, and reproductive tracts.1−4 Bovine submaxillary mucin (BSM) is secreted in the saliva that lines teeth, tongue, and palate, and interacts with food, external liquids, and air. General structural features of mucins include a long central domain rich in proline, threonine and serine (PTS domain) and densely grafted with anionic and hydrophilic carbohydrate chains. The C- and N-terminals are not glycosylated and contain many cationic and hydrophobic residues. The glycosylation level of the PTS domain reaches 70−85% of the total molecular weight in BSM and sialic acid accounts for as much as 30% of the molecular weight.5,6 The mucus coating provides a protective barrier against adsorption of unwanted biomolecules, bacteria, and particles, reduces friction, and prevents wear of the underlying tissues. Mucins abundantly adsorb onto nonbiological hydrophobic surfaces7,8 and, to a lesser extent, negatively charged surfaces9 by tethering the end domains. Adsorbed mucins coatings display interesting surface © 2015 American Chemical Society

Received: February 10, 2015 Revised: March 24, 2015 Published: March 25, 2015 4524

DOI: 10.1021/acs.langmuir.5b00548 Langmuir 2015, 31, 4524−4533

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Langmuir gastric mucin,15 porcine gastric mucin,16 lubricin,17−19 and BSM.5,13,20 In this study, we demonstrate that BSM forms highcoverage and stable layers on mica surface, and reduces adhesion and layer plasticity effects commonly observed for low-coverage mucin or polymer films. Acidification of the aqueous medium was taken as a means to alter the electrostatic interactions of the numerous anionic charged residues of the PTS domain, thereby revealing their influence on molecular conformation and force generation. Acidification is exemplificative of the unpredictable environmental changes to which BSM and other mucins are commonly exposed (e.g., in the contact with food). Understanding how mucins respond to these changes may help developing more resistant and versatile coatings for engineering applications.

Chirascan spectrophotometer (Applied Photophysics Ltd., Surrey, U.K.). Far-UV CD spectra were recorded from 195 to 245 nm with step size of 1 nm and bandwidth of 1 nm and near-UV CD spectra were recorded from 400 to 260 nm with step size of 2 nm and bandwidth of 0.8 nm. Temperature was maintained at 25 °C using a CS/PCS single cell Peltier temperature controller (Applied Photophysics Ltd., Surrey, U.K.). The presented data represent three independent measurements, each averaged over three scans. 2.5. Atomic Force Microscope (AFM). We used a MultiMode AFM with a Nanoscope IIIa controller (Bruker-Veeco, Santa Barbara, CA), RTESPA/MPP-11120-10 probes (from the same producer, with rectangular cantilever, silicon tips, 200−400 kHz resonance frequency and 20−80 N/m spring constant), and intermittent contact mode (tapping) in air to perform high-resolution imaging of single molecules and aggregates. BSM was adsorbed on mica surfaces from very dilute solution in neutral PBS, with concentration lower than 0.01 mg/mL. Before adsorption, the mica surfaces were immersed in 2 mM solution of NiCl2 in purified water for 30 s, rinsed with purified water, and dried with nitrogen. Such treatment favors the adsorption of isolated mucins parallel (flat) to the surface due to the attraction between the negatively charged PTS domain and cationic surface sites created by the adsorption of Ni2+ ions to native anionic sites of the mica surface.23,24 2.6. Surface Force Apparatus (SFA). Normal force measurements between mica surface bearing adsorbed layers of BSM were obtained using a SFA (Mark III from Surforce LLC). A detailed description of the technique can be found in ref 25. Atomically smooth sheets were cleaved from thick mica plates (from JBG Metafix, France), coated with a semireflective Ag layer and glued with the silvered side onto half-cylindrical glass lenses. The surfaces were mounted in the SFA in a crossed cylinder geometry, which can be approximated as a sphere facing a plane. The surface separation D at the contact point and the radius of curvature R = 1.8−2.2 cm were measured with an accuracy of 0.5 nm and 0.01 cm, respectively, using an optical interferometric technique based on the measurement of the wavelengths that emerge from constructive multiple reflections between the Ag layers.26 We also measured the average refractive index n of the medium between the surfaces at the contact position with an accuracy of about 0.05. From the n(D) curve, we determined the surface coverage Γ of BSM on mica using the formula:27

2. MATERIALS AND METHODS 2.1. BSM and Purification. Bovine submaxillary mucin (BSM; type I-S, M3895) was purchased from Sigma-Aldrich (Brøndby, Denmark). In order to minimize nonmucin impurities present in commercial mucins, anion exchange chromatography was employed to further purify the BSM samples as described in details in ref 14. Briefly, BSM was dissolved in Na-acetate buffer (10 mM Na-acetate, 1 mM EDTA, pH 5.0) at 4 °C to a final concentration of 10 mg/mL, filtered through a 5 μm sterile filter (Pall Corporation, Cornwall, U.K.), and fractionated on a High Load 16/26 Q Sepharose High Performance anion exchange column (GE Healthcare Life Sciences) by using Naacetate buffer as eluent. Purity of the obtained BSM fractions was analyzed with SDS-PAGE that revealed nearly undetectable level of impurities.14 The fractions containing BSM were pooled, dialyzed against water, freeze-dried and kept at −20 °C until use. All the experimental results presented in this study were carried out exclusively with purified BSM. 2.2. Chemicals and Buffer Solutions. BSM solutions at concentrations 0.15−2.2 mg/mL were obtained by thawing lyophilized BSM at room temperature and dissolving in filtered phosphate buffered saline (PBS, from Sigma-Aldrich) with purified water (Millipore Q from Millipore, resistivity > 18.2 MΩ·cm). Neutral solution had pH 7.4 and Debye length λ = (εkT/I) 1/2 ≈ 1 nm, where I = ΣCiqi2 and ε is the dielectric permittivity of water, and qi and Ci are the charge and molarity of each ionic species.21 Acid solutions with pH 2.6−2.9 were obtained by adding small amounts of HCl to neutral PBS, or a volume of acetic acid (AcOH) equal to the volume of neutral PBS. In both cases, the relative variation of the Debye length was δλ/λ ≈ 1/2δI/I ≈ δCPBS/CPBS ≈ 1/4, that is, λ varied by less than 1 nm. 2.3. Dynamic Light Scattering (DLS). DLS was employed to characterize the hydrodynamic diameter, dh, of BSM molecules in different pH environment using Malvern Zetasizer Nano ZS two angle particle and molecular size analyzer (Malvern Instruments, Worcestershire, U.K.). Noninvasive back scatter (NIBS) technique was employed, using a He−Ne laser light source (wavelength λ = 633 nm), scattering angle θ = 173°, and scattering vector q = 4πn sin(θ/ 2)/λ = 0.026 nm−1. The instrument provided the normalized timeautocorrelation function of the scattered intensity, g(t). For molecules with radius of gyration rg such that qrg ≤ 1, DLS probes the translational diffusion of dissolved molecules. The correlation function is g(t) ∝ ρS(qrg) exp(−cq2t), where ρ is the density of molecules in the scattering volume, S is the dynamic form factor, and c is the diffusion coefficient.22 The Malvern Zetasizer software (version 7.02) was used to determine the distribution of hydrodynamic diameter, dh = kT/ (3πηc), in terms of the diffusion coefficient c, where η is the viscosity of water. Prior to the preparation of BSM solutions, buffer solutions were filtered with 0.22 μm pore radius filters. Disposable cuvettes (PMMA, Plastibrand) were employed for DLS measurements. The concentration of samples was 2 mg/mL. All measurements were done at 25 °C. 2.4. Circular Dichroism (CD) Spectroscopy. Far-UV and nearUV CD spectroscopies were employed to characterize the secondary and tertiary structure of BSM in different pH environment using a

n = nb + (2Γ/D)dn/dC

(1)

where nb = nw = 1.333 is the refractive index of the bulk protein solution, close to the value nw of water, and dn/dC = 0.150 cm3/g is the refractive index variation as a function of BSM mass concentration.28 To measure the normal force F, one of the surfaces was attached to the free end of a double cantilever spring with elastic constant K = 240 N/m or 950 N/m, whose fixed end was attached to a motorized actuator. The force could be determined with an accuracy of about 10−6 N by monitoring the spring deflection. Experiments with surfaces having different radii R were compared by considering the normalized force F(D)/R which, according to Derjaguin relation, does not depend on R.21 The distance D = 0 was defined by the set of wavelengths recorded at mica−mica contact in dry air (before adsorbing BSM). The surfaces were then separated and a droplet BSM solution with volume 50−100 μL was inserted by capillarity. We let the BSM adsorb from neutral buffer solution for 10−15 h while keeping the surfaces well separated. Acid solutions infiltrated the Agmica interface leading to a progressive detachment of the mica sheet from the glass supports. Therefore, we limited the adsorption time of BSM from acid solution to 5 h. Solution conditions were changed in situ, without dismounting the surfaces, by adding 1−3 mL of protein-free buffer solution to the droplet while aspirating with a pipet the excess liquid that spilled out from the surfaces. For each set of surfaces and after changing the solution conditions, we considered at least two different contact positions. For each contact position, we measured the force curve F vs D at least twice. Before SFA experiments, BSM solutions were centrifuged for 3 min at 4000 rpm to remove aggregates and 4525

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Langmuir contaminant particles. Adsorption and force measurements were done at a fixed temperature of (25 ± 1) °C.

3. RESULTS AND DISCUSSION 3.1. DLS: Hydrodynamic Size of BSM in Bulk Solution. The distribution of hydrodynamic diameter, dh, of BSM molecules in pH 7.4 and 2.6 buffer solutions determined by DLS is plotted according to the intensity in Figure 1. At pH 7.4,

Figure 2. Far-UV CD spectra for 2 mg/mL BSM solution in PBS at pH 7.4 and 2.6. The inset shows the near-UV CD spectra.

slight positive shoulder at 220 nm, in consistence with previous studies on BSM14 and ovine submaxillary mucin (OSM).31 At pH 2.6, no major changes in the far-UV CD spectrum occurred, except that the overall intensity of the negative peak was slightly reduced. As far-UV CD spectra of BSM are determined mainly from the central glycosilated region, this indicates that the conformation of PTS region is largely maintained upon lowering the pH from 7.4 to 2.6, except for a slight weakening of coiling. Meanwhile, no discernible peak was observed in the near-UV CD spectra, at both pH 7.4 and 2.6 (Figure 2, inset), indicating that a distinct three-dimensional folding was also absent for both pH values. 3.3. Contour Length and Conformation of Single BSM Molecules. Figure 3 shows an AFM image of BSM molecules adsorbed on a Ni-treated mica surface. The molecules were adsorbed parallel to the surface and appeared as linear objects. Local departures from the linear shape and small globular features were frequently observed along the molecule axis (Figure 3b). These features were randomly distributed along the molecules and at their ends, or were completely absent. In Figure 4, BSM molecules were aligned by the flow created upon rinsing with purified water and subsequent drying with nitrogen. In this case, molecules were straightened and flattened. These observations suggest that nonlinear and globular regions corresponded to bends, coils and twists of the randomly coiled protein chain (Figure 3b), that could not be resolved by AFM and were unfolded by flow. The height of flat (globule-free) molecular segments was uniform, h = (3.4 ± 1.1) Å, and consistent with the value h < 1 nm reported for other mucins imaged in air.24 The cross-sectional width of the molecules, w = 7−8 nm, was much larger than h due to shape convolution effects between the molecule and the spherical AFM tip with radius ∼ 10 nm. Flat isolated molecules with uniform height h were always linear and unbranched (Figure 4b), with contour lengths varying over a wide range from a few tens of nanometers to about 1 μm. These observations indicate that BSM molecules aggregated mainly by pairwise association of their ends (as opposed to branched or star-like aggregation). Branching was always associated with nonflat globular features with height 7− 12 Å that could not be distinguished from random crossing or overlapping of the molecules (Figure 3b).

Figure 1. Distribution of the hydrodynamic diameter dh of BSM as a function of DLS intensity (2 mg/mL in PBS buffer at pH 2.6 and 7.4).

a single peak was observed in the range dh = 10−200 nm, corresponding to more than 99% of the entire peak area. The maximum of intensity was measured for a diameter dm = (46 ± 1) nm and the Z-average value was dz = (35 ± 2) nm. At pH 2.6, the intensity maximum shifted to a smaller diameter dm = (41 ± 2) nm and a secondary peak appeared for large diameters dh = 150−1000 nm. Accordingly, the Z-average diameter increased to dz = (44 ± 1) nm. These results are also summarized in Table 1. Given that the contraction of dm and Table 1. Parameters Describing the Conformation of BSM Molecules in Solution (dm, dz) and after Adsorption on Mica (H, Γ, ρ, A, d) from 2.0 mg/mL Solution in PBSa neutral, X1 dm (nm) dz (nm) H (mg/m2) Γ (mg/m2) ρ (mg/mL) A (mN/m) d (nm)

35 46 60 2.4 40 22 20

± ± ± ± ± ± ±

2 1 5 0.2 7 2 2

acid, X2

X2/X1

± ± ± ± ± ± ±

1.26 0.89 1.58 3.96 2.50 1.50 1.30

44 41 95 9.5 100 33 26

1 2 5 0.5 13 2 2

a

X1 and X2 indicate the parameter value for neutral and acid solution, respectively.

appearance of the secondary peak occurred in parallel at low pH, both changes can be related to the protonation of polyanionic BSM at acidic pH and the reduction of intra- and intermolecular electrostatic repulsions between negatively charged residues, particularly in the PTS domain. 3.2. Secondary and Tertiary Structure of BSM Molecules. CD spectroscopy has extensively been employed as a tool to investigate the conformation of globular proteins,29,30 yet to a much lesser extent for mucins.4,31 As presented in Figure 2, the far-UV CD spectra obtained from BSM at pH 7.4 is indicative of random coil structure with a characteristic strong negative band at around 200 nm and a 4526

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Figure 3. (a) AFM image of BSM molecules adsorbed on Ni-treated mica from dilute neutral PBS solution. (b) Magnification showing recurring morphological features. The height relative to the mica substrate is color-coded as shown by the color bar to the right.

Figure 4. (a) AFM image of BSM molecules adsorbed on a Ni-treated mica from dilute neutral PBS solution. Molecules were aligned upon rinsing/ drying the surface. (b) Individual molecules and aggregates adsorbed in a flat conformation. The horizontal scale bar is 50 nm in all images. The vertical color bars show the height scale.

Figure 5. Distribution of contour length l for isolated BSM molecules. Dark gray indicates flat molecules (without globular features) straightened by fluid flow. Medium gray indicates flat molecules (straightened or not). Light gray indicates molecules with at most one globule. Vertical dashed lines are located at values of l multiple of l0 = 55 nm. The solid line is the distribution of hydrodynamic diameter dh measured by DLS in neutral solutions (from Figure 1).

the fact that AFM could not resolve the “hidden” length involved in bends, coils, and globules that frequently decorated the BSM molecule (Figures 3b and 4a). We limited the analysis to molecules that showed variations of height and width smaller

Figure 5 shows the distribution of contour length l for isolated BSM molecules, that is, molecules that did not overlap any other molecule and were entirely exposed (with both ends visible) within an AFM image. The accuracy of l was limited by 4527

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Figure 6. (a) Normalized force F/R as a function of the mica−mica distance D after adsorption of BSM from 1.8 mg/mL solution in PBS with pH 7.4. Different symbols indicate different experiments or contact positions. Each color represents a different approach/retraction cycle. Filled and open symbols indicate surface approach and retraction, respectively. (b) Semilogarithmic plot showing the exponential decay of F for large D. 2H is the onset of the repulsion. (c) Refractive index n as a function of D. The dotted and solid lines indicate respectively the values expected in the absence of adsorption (n = nb = 1.333) and for the indicated surface coverage Γ. (d) Force curve obtained after rinsing with protein-free HCl/PBS with pH 2.9. The shaded area contains all data points obtained before rinsing.

3.4. Surface Force Measurements. Figure 6a shows the force F, normalized by the radius of curvature R of the surfaces, as a function of the mica−mica separation distance D after adsorption of BSM from a 1.8 mg/mL solution in neutral PBS. The force became repulsive and consistently larger than the measurement error of 0.3 mN/m when the adsorbed BSM layers touched each other at a distance D = 2H = (120 ± 10) nm, and increased as D was decreased. The layer thickness was approximately equal to H = (60 ± 5) nm. The same force curve F(D) was measured upon approaching and retracting the surfaces over many approach/retraction cycles. The absence of hysteresis or viscous response indicates that the measurements were done under equilibrium conditions. BSM layers could repeatedly withstand normalized loads as high as 30 mN/m and layer deformations δ = (2T − Dmin)/2 = (120−36)/2 nm = 42 nm without being permanently altered, corresponding to an average pressure P = F/S ≈ 60 kPa over the contact area S = 2πRδ ≈ 4600 μm2, where R ≈ 2 cm. The force curve increased almost exponentially as D was decreased from 125 nm to about 35 nm: F/R=A e−D/d with a decay length d = 20 nm and prefactor (amplitude) A = 22 mJ/m (Figure 6b). As D was further decreased, F increased faster than exponentially. The refractive index n increased above the bulk

than 2h and 2w, respectively, and molecules showing at most one globular feature, that is, a region with height and width larger than h and w, respectively. The length distribution showed a main peak in the range l = 40−180 nm with a maximum at lm = (95 ± 10) nm and a long tail extending beyond 300 nm. Both lm and the peak width were about twice as large as those measured by DLS for the distribution of hydrodynamic diameter dh (solid line in Figure 5). The distribution of contour length also showed local maxima at multiples of the length l0 ≈ 55 nm (Figure 5). These peaks were more pronounced for molecules without globular domains and molecules straightened by fluid flow, that is, when the accuracy on l was higher. These results suggest that a molecular species with contour length close to l0 was either more abundant or more active in forming pairwise end-to-end aggregates. We point out that the distribution of l also included molecules whose lengths were clearly not multiple of l0, such as the one shown at the top of Figure 4b (l = 97 nm). It is not clear whether these species were molecular fragments created during extraction and purification, distinct mucin types that were not differentiated by the purification process, or BSM variants encoded by the BSM gene.4,32 4528

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Figure 7. (a) Normalized force F/R measured as a function of the mica−mica distance D for neutral BSM/PBS solutions at different concentrations, C, after adsorption (●/○, ◆/◇) and after rinsing (■/□, ▲/△) with protein-free acid AcOH/PBS at pH 2.6. (b) Semilogarithmic plot showing the exponential decay of F for large D. The shaded area in (a) and dashed line in (b) represent the data of Figure 6.

Figure 8. (a) Normalized force F/R as a function of the mica−mica distance D after adsorption of BSM from 2.0 mg/mL acid HCl/PBS solution and after rinsing with neutral PBS. In all figures, the shaded area and dashed line represent the data obtained after adsorption from neutral BSM/PBS solutions. (b) Semilogarithmic plot of the force curves showing the exponential decay of F(D). (c) Refractive index n as a function of D. Solid lines correspond to the values calculated for the indicated values of surface coverage Γ. (d) Comparison of the forces obtained after rinsing with neutral PBS (circles and squares) and after rinsing again with acid HCl/PBS (triangles).

value nb as D was decreased (Figure 6c). A fit of the n(D) curve with eq 1 gave a surface coverage Γ = (2.4 ± 0.2) mg/m2. The

protein mass density in the adsorbed layers was ρ = Γ/H = 40 mg/mL, that is 24 times larger than that in the bulk (Table 1). 4529

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the same mass as those in PTS domain and the percentage of glycans in the total molecular weight is 50−80%, the total MW of BSM can be estimated from AFM images to be 200−500 kDa. 4.2. Nanomechanics of Adsorbed BSM Layers. BSM has an isoelectric point IEP ≈ 3 and is negatively charged at neutral pH due to the abundance of sialic acid in the PTS domain. BSM also contains many positively charged and hydrophobic residues in the end domains. Since the mica surface is also negatively charged in water (IEP ≈ 3),39 the adsorption of BSM on mica at neutral pH is likely due to binding of the end domains (Figure 9).5,13,20 The surface

Adsorption from more concentrated (2.2 mg/mL) or more dilute (0.15 mg/mL) neutral BSM/PBS solutions produced similar force curves with layer thickness H, amplitude A and decay length d decreasing as the concentration decreased (Figure 7). Rinsing the surfaces with protein-free neutral PBS did not alter the force profiles, showing that BSM molecules were not desorbed upon rinsing and unadsorbed molecules could be flushed away upon rinsing without affecting the normal forces (data not shown).5 Moreover, when a BSM layer adsorbed from neutral PBS was rinsed with protein-free acid HCl/PBS (Figure 6d) or AcOH/ PBS (Figure 7), the force curves did not show any significant change. Adsorption from low pH, despite the shorter adsorption time, produced layers with greater surface coverage Γ, larger thickness H, density ρ, prefactor A of the exponential force and a relatively small increase of the decay length d (Figure 8a−c and Table 1). After rinsing with protein-free neutral PBS, all parameters decreased due to desorption of BSM from the layers. The force became less repulsive (Figure 8a) but the surface coverage remained larger (Figure 8c) than for BSM layers adsorbed from neutral solution, suggesting a different layer structure. When the layers were rinsed again with proteinfree acid HCl/PBS, the force did not change (Figure 8d).

Figure 9. Schematic representation of BSM molecules adsorbed on mica from PBS solution. l and a are the average contour length and persistence length of a single molecule. Molecules are grafted to the substrate by their ends at an average distance s from each other. H is the height of the adsorbed layer.

4. DISCUSSION 4.1. Single Molecules. Because of the heavy glycosylation of the PTS domain, BSM molecules can be considerably more rigid than unglycosylated proteins. Strongly hydrated and negatively charged sugar groups of the PTS domain repel each other by steric and electrostatic interactions that restrict the range of orientation angles between consecutive amino acids along the polypeptide chain. As a consequence, mucins are often described as “wormlike” semiflexible polymer chains with contour length l and persistence length a approaching the limit l/a ≈ 1.31,33−36 In fact, a is of the order of 10 nm, much larger than the value a ≈ 0.4 nm for nonmucin, unstructured proteins.24,31,34 For l/a ≫ 1, a wormlike chain forms an ideal random coil with radius of gyration rg ≈ (al/3)1/2 ≪ l (neglecting the chain self-avoidance), whereas for l/a ≪ 1 the chain behaves as a rigid rod with rg ≈ (l2/12)1/2 ≈ l.37 For all values of l/a, the ratio dh/2rg is in the range 1−3.5, where the lower bound corresponds to the rigid rod limit.38 Our findings that both the maximum point (dm) and width of the distribution of dh (Figure 1) are about half the corresponding values in the distribution of l (Figure 5) indicates that BSM should be considered as a stiff chain with l/a ≥ 1 and dh ≈ 2rg ≈ l, rather than a polymer-like random coil with l/a ≫ 1 and rg ≪ l. The hydrodynamic diameter and molar weight of BSM monomers have previously been reported to be dh = 40−76 nm and MW = 0.8−2.0 MDa, respectively.6,21 While the diameter compare well with the values of dm and dz obtained in the present study (Table 1), MW seems too high for the short contour length measured by AFM, l < 300 nm (Figure 5). Indeed the length of the PTS domain increases approximately as l = pN where N is the number of monomeric residues in the domain prior to glycosylation and p ≈ 0.25 nm/residue is the typical pitch of the wormlike chain.31 If we consider an average molar mass of 120 g/mol for amino acids in the PTS domain and N = l/p ≈ 100/0.25 = 400, the molecular weight of the PTS domain is about 50 kDa, excluding carbohydrate chains. If we further suppose that the peptides in the end domains have

coverage Γ is higher than the values measured on silica surfaces in saline solution,28,40 despite silica having a higher surface density of negative charges.41,42 This suggests that nonelectrostatic interactions also contribute to BSM-mica adsorption interaction.43 The repulsive force between two surfaces coated with endgrafted polymer molecules can be described with two different models depending on the ratio 2rg/s between the radius of gyration in solution and the grafting distance s on the surface (Figure 9). When 2rg/s < 1, the layer is dilute and the force is given by21 F e−D / rg = A e−D / d = 72πkT 2 R s

(2)

for distances such that D/2rg = 1−4. For 2rg/s > 1, the layer is dense (or “semidilute”) and the lateral interaction between adsorbed molecules forces them to stretch along the substrate normal, forming a “brushlike” layer. The force is given by F H e −π D / H , = A e−D / d = 64πkT R s3

with H = l(a /s)2/3 (3)

for distances D/2H = 0.2−0.9, where H is the layer thickness. On one hand, BSM layers adsorbed from neutral solution (concentration 1.8−2.2 mg/mL) produced an exponentially repulsive force with decay length comparable to the gyration radius, d = 20−26 nm ≈ dm/2 (Figures 6b and 7b, and Table 1), in agreement with the dilute layer model (eq 2). On the other hand, BSM display a rigid rod-like conformation in solution and may sustain such conformation after being adsorbed onto mica surface, even in dilute regime. Thus, primarily owing to its unique stiff conformation rather than grafting density, BMS may stretch and form adsorbed layers that are semidilute and brushlike (Figure 9). Indeed, the layer 4530

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higher density ρ (higher volume fraction ϕ) of BSM molecules in the layer (Table 1). The density increase may also be due to stronger attractive interactions between BSM and mica at low pH, due to charge neutralization and decreased repulsion of negative charges of both BSM and mica. However, the density increase was observed also for BSM adsorbing on uncharged apolar surfaces at low pH, where BSM-mica interactions are not expected to be pH-dependent.28 Therefore, the density increase appears to be driven mainly by intersegment interaction. Inside the densely packed layers, end-to-end association (Figure 5) is much more likely to occur than in solution, increasing the contour length l of the worm-like chains. The volume fraction ϕ inside an adsorbed polymer layer is expected to depend on the excluded volume per segment, which does not depend on l.37 Therefore, a pH-dependent increase of l due to end-to-end association cannot explain the density increase at low pH. On the other hand, acidification favors aggregation in solution (Figure 1) and may induce the formation of long chains at the surface, increasing the thickness of the layer: H ∝ l (eq 3). Desorption was observed upon rinsing with neutral solution a layer adsorbed from acid solution (Figure 8a and b). In this case, electrostatic repulsion between PTS domains was restored and osmotic pressure inside the layer increased to a point that BSM−mica adsorption interactions were not sufficient anymore to stabilize the layer structure. BSM−mica interaction may also become more repulsive as the pH is increased and negative charges are restored on both BSM and mica. However, as previously noted, changes in adsorption density can be also induced for BSM layers adsorbed on uncharged surfaces.28 It is noteworthy that rinsing with acidic solution a layer that was stable at neutral pH (Figures 6d, 7, and 8d) did not produce any desorption because rinsing reduced the electrostatic repulsion and osmotic pressure. Moreover, the normal forces F remained almost unaltered after acidification (Figures 6d, 7, and 8d). In fact, F depends on pH via the grafting distance s and contour length l (eq 2 and 3), since the persistence length a and gyration radius rg do not significantly change upon acidification. Rinsing with protein-free acid solution did not produce desorption (i.e., s remained constant) and could not induce chain elongation (l can increase only when proteins are added to the layer from the solution) or chain shortening/fragmentation (acidification enhances association, Figure 1). Therefore, the force F could only remain constant upon acidification of a layer adsorbed at neutral pH.

thickness H is comparable to the contour length l and the decay length is d ≈ H/π ≈ l/π ≈ 30 nm, as in eq 3. Therefore, both the dilute and brush layer model can be fitted to the force curves. The prefactor A = 22 mN/m of the normalized force can be obtained with s = 6.4 nm in eq 2, or s = 13 nm and H = 60 nm in eq 3. The molecular weight estimated from the relationship MW = NAΓs2 with the experiment value Γ = 2.4 mg/m2 (Table 1) is 60 kDa in the dilute layer case and 250 kDa in the brush layer case. Therefore, the brushlike description of the adsorbed layer is more consistent with the molecular weight estimated from the morphology imaging by AFM (subsection 4.1). For both the dilute and brush layer models, F increases faster than an exponential for D/rg ≪ 1 or D/H ≪ 1, respectively, as observed in all force curves. When BSM was adsorbed from dilute solution, H = 23 nm was comparable to dh but the decay length was d < dh/2 (Figure 7), in contrast to what expected for a dilute layer (eq 2). Moreover, the force showed a second exponential regime for D < 20 nm with a shorter decay length of about 4 nm. Most likely, the conformation of BSM molecules in the dilute layers varied during surface approach, e.g. molecules became more parallel to the surface, thereby changing the mechanical response to the external compressive load. 4.3. Effect of Acidification. At pH < 3, more than half of the negative charges in the PTS domains are neutralized. The bending rigidity and persistence length a are expected to decrease as a result of the reduced electrostatic repulsion between neighboring negatively charged sugars along the protein chain. Therefore, the hydrodynamic radius is expected to decrease. While this was indeed observed, the variation δdm/ dm ≈ −0.11 was small (Figure 1 and Table 1). In the limit of l/ a ≈ 1, this variation corresponded to a comparable decrease of the persistence length δa/a ≈ δdm/dm ≈ −0.1. Such small variation indicates that steric hindrance between sugar chains is more important than electrostatic repulsion in determining the persistence length a at the physiological salinity of our solutions, in agreement with similar results obtained from previous studies on BSM1 and OSM.31 It is worth noting here that the Debye length, λ ≈ 1 nm, may be shorter than the average spacing between neighboring charged residues along the polypeptide chain, so that electrostatic interactions between these residues may be effectively screened both at low and neutral pH. The conformation of isolated chains molecules and stability of adsorbed layers of chains depend on intramolecular interactions between nonconsecutive segments (far from each other along the same chain) and intermolecular interactions between segments belonging to different chains. These interactions modify the effective excluded volume per segment that plays a central role in Flory theory of real polymer chains and are commonly referred to as “excluded volume” effects.37 Within an isolated wormlike chain molecule with l/a ≈ 1, such as BSM in solution, the number of “contacts” between nonconsecutive segments is relatively small and intersegment interactions can be neglected.37 On the other hand, the protein mass density ρ inside adsorbed BSM layers (Table 1) is much larger than the density MW/(NAl3) ≈ 1 mg/mL (“overlap” density37) inside the volume pervaded by an isolated wormlike BSM molecule in solution, that is of the order of l3. Therefore, the probability of contact is high and intersegment interactions may play an important role. Indeed adsorbing from acid solution, that is, neutralizing negative charges and reducing electrostatic repulsion between acidic PTS segments, led to

5. CONCLUSIONS Understanding the molecular-scale mechanisms underlying the surface properties of mucins such as hydration,7 mucoadhesion,10 and lubrication8,11,12 is crucial for the biomimetic development of biocompatible, environment-friendly polymeric materials and coatings for water-based applications.44−46 In this study, we investigated the relation between the structure and properties of single BSM molecules, and the nanomechanics of dense BSM layers adsorbed on charged surfaces such as mica. Using highly purified BSM was essential to minimize the influence of nonmucin impurities. Complementary observations by DLS, CD and AFM revealed that BSM molecules behave as rigid “wormlike” polypeptide chains lacking a defined protein structure, due to the heavy glycosylation of the central PTS domain. The persistence length of the domain was not affected by acidification at physiological salinity, indicating that the stiffness of the PTS domain is mainly due to steric 4531

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hindrance between acidic side sugar chains, rather than their mutual electrostatic repulsion. Using the SFA study, we have shown that BSM readily adsorbs on mica with a density much higher than the overlap concentration. Strong, long-ranged, reversible (elastic) repulsive forces are generated between BSM-coated surfaces, which can be modeled using current theories for polymer-like molecules. Intersegment interactions (i.e., excluded volume effects) were found to play a significant role during BSM adsorption, leading to a higher adsorbed mass density at low pH due to reduced electrostatic repulsion between PTS domains. Conversely, increasing the pH after adsorption restored electrostatic repulsion and induced desorption. Remarkably, acidification of the layers adsorbed from neutral solution did not produce desorption or variations of the repulsive forces. Such ability of BSM to remove adhesion and prevent the direct contact between two surfaces during compression, and the stability against acidification can be linked to the native function of BSM in the oral cavity as a surface protective and lubricating agent at work in variable pH conditions.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

The financial support from European Research Council (ERC) (Funding Scheme: ERC Starting Grant, 2010, Project Number 261152) is acknowledged. We thank Dr. Maria De Santo at the University of Calabria for helping us with the AFM imaging of single molecules.

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