Structural and Mechanical Properties of Thin Films of Bovine Submaxillary Mucin versus Porcine Gastric Mucin on a Hydrophobic Surface in Aqueous Solutions.

The structural and mechanical properties of thin films generated from two types of mucins, namely, bovine submaxillary mucin (BSM) and porcine gastric mucin (PGM) in aqueous environment were investigated with several bulk and surface analytical techniques. Both mucins generated hydrated films on hydrophobic polydimethylsiloxane (PDMS) surfaces from spontaneous adsorption arising from their amphiphilic characteristic. However, BSM formed more elastic films than PGM at neutral pH condition. This structural difference was manifested from the initial film formation processes to the responses to shear stresses applied to the films. Acidification of environmental pH led to strengthening the elastic character of BSM films with increased adsorbed mass, whereas an opposite trend was observed for PGM films. We propose that this contrast originates from that negatively charged motifs are present for both the central and terminal regions of BSM molecule, whereas a similar magnitude of negative charges is localized at the termini of PGM molecule. Given that hydrophobic motifs acting as an anchor are also localized in the terminal region, electrostatic repulsion between anchoring units of PGM molecules on a nonpolar PDMS surface leads to weakening of the mechanical integrity of the films.

Molecular Structure and Equilibrium Forces of Bovine Submaxillary Mucin Adsorbed at a Solid-Liquid Interface.

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.

Hydrophobins as aqueous lubricant additive for a soft sliding contact.

Two type II fungal hydrophobins, HFBI and FpHYD5, have been studied as aqueous lubricant additive at a nonpolar, compliant sliding contact (self-mated poly(dimethylsiloxane) (PDMS) contact) at two different concentrations, 0.1 mg/mL and 1.0 mg/mL. The two hydrophobins are featured as non-glycosylated (HFBI, m.w. ca. 7 kDa) vs glycosylated (FpHYD5, m.w. ca. 10 kDa) proteins. Far UV CD spectra of the two hydrophobins were very similar, suggesting overall structural similarity, but showed a noticeable difference according to the concentration. This is proposed to be related to the formation of multimers at 1.0 mg/mL. Despite 10-fold difference in the bulk concentration, the adsorbed masses of the hydrophobins onto PDMS surface obtained from the two solutions (0.1 and 1.0 mg/mL) were nearly identical, suggesting that a monolayer of the hydrophobins are formed from 0.1 mg/mL solution. PDMS-PDMS sliding interface was effectively lubricated by the hydrophobin solutions, and showed a reduction in the coefficient of friction by as much as ca. two orders of magnitude. Higher concentration solution (1.0 mg/mL) provided a superior lubrication, particularly in low-speed regime, where boundary lubrication characteristic is dominant via 'self-healing' mechanism. FpHYD5 revealed a better lubrication than HFBI presumably due to the presence of glycans and improved hydration of the sliding interface. Two type II hydrophobins function more favorably compared to a synthetic amphiphilic copolymer, PEO-PPO-PEO, with a similar molecular weight. This is ascribed to higher amount of adsorption of the hydrophobins to hydrophobic surfaces from aqueous solution.

Conformation of bovine submaxillary mucin layers on hydrophobic surface as studied by biomolecular probes.

In the present study, the conformational changes of bovine submaxillary mucin (BSM) adsorbed on a hydrophobic surface (polystyrene (PS)) as a function of concentration in bulk solution (up to 2mg/mL) have been investigated with biomolecular probe-based approaches, including bicinchoninic acid (BCA), enzyme-linked immunosorbent assay (EIA), and enzyme-linked lectin assay (ELLA). The conformation and hydrodynamic diameter of highly purified BSM molecules, as characterized by circular dichroism (CD) spectroscopy and dynamic light scattering (DLS), respectively, showed a slight, yet gradual coiling and compaction in response to the increase in BSM concentration in bulk solution. Adsorbed masses of BSM onto hydrophobic surface, as probe by BCA, showed a continuously increasing trend up to 2mg/mL. But, the signals from EIA and ELLA, which probe the concentration of available unglycosylated C-terminals and the central glycosylated regions, respectively, showed complicated non-linear responses with increasing surface concentration. The results from this study support the conventional amphiphilic, triblock model of BSM in the adsorption onto hydrophobic surface from aqueous solution. The biomolecular probe-based approaches employed in this study, however, provided further details on the conformational changes of BSM on surface, in particular the accessibility of glycosylated and unglycosylated domains with increasing surface concentration.

A simplified chromatographic approach to purify commercially available bovine submaxillary mucins (BSM).

In this study, a simple purification protocol is developed to reduce the bovine serum albumin (BSA) content in commercially available bovine submaxillary mucin (BSM). This involved purification of the BSM by one-column anion-exchange chromatography protocol resulting in BSM with greatly reduced BSA content and homogeneously distributed size, and in a high yield of ∼43% from BSM as received from the manufacturer. The purity and composition of commercially acquired BSM were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry, which verified that BSA is the most abundant nonmucinous protein component. The purification effect was evident from a significantly altered circular dichroism (CD) spectrum of BSM after anion-exchange chromatography.

Thermostability of bovine submaxillary mucin (BSM) in bulk solution and at a sliding interface.

Thermostability of bovine submaxillary mucin (BSM) was studied in terms of its structure, hydrodynamic size, surface adsorption, and lubricating properties in the temperature range of 5-85°C. The overall random coil structure of BSM showed a gradual loosening with increasing temperature as characterized by circular dichroism (CD) spectroscopy, but this change was fully reversible upon lowering temperature. Extended heating up to 120 min at 80°C did not make any appreciable changes in the structure of BSM when it was cooled to room temperature. The hydrodynamic size of BSM, as studied by dynamic light scattering (DLS), showed a slight increase after heating at high temperature (80°C). Optical waveguide lightmode spectroscopy (OWLS) studies showed facile adsorption of BSM onto poly(dimethylsiloxane) (PDMS) surface (>180 ng/cm(2)) at room temperature due to its amphiphilic characteristics. Adsorbed mass of BSM was noticeably reduced after heating at 80°C, possibly resulting from its aggregation. BSM showed excellent lubricity at self-mated sliding contacts between PDMS at room temperature or lower (friction coefficient≈0.02), even when BSM solution was pre-heated up to 120 min at 80°C. Gradual degradation of lubricity of BSM was observed with increasing temperature, but it was also reversibly recovered with decreasing temperature. Structural and functional stability of BSM against heating is proposed to originate from heavy glycosylation and lack of higher degree of protein structure in BSM.

Mechanics and dynamics of triglyceride-phospholipid model membranes: Implications for cellular properties and function.

We demonstrate here that triolein alters the mechanical properties of phospholipid membranes and induces extraordinary conformational dynamics. Triolein containing membranes exhibit fluctuations up to size range of 100µm and with the help of these are e.g. able to squeeze through narrow passages between neighbouring structures. Triolein-phosphatidylcholine membranes were found to have bending rigidity significantly lower than that of corresponding pure phosphatidylcholine membrane. Moreover, the triolein containing membranes were found to be reluctant to fuse, which is in good accordance with larger lamellar distances observed in the TOPOPC membranes. These findings suggest repulsion between adjacent membranes. We provide a comprehensive discussion on the possible explanations for the observed mechanics and dynamics in the TOPOPC system and on their potential cellular implications.

Triglyceride blisters in lipid bilayers: implications for lipid droplet biogenesis and the mobile lipid signal in cancer cell membranes.

Triglycerides have a limited solubility, around 3%, in phosphatidylcholine lipid bilayers. Using millisecond-scale course grained molecular dynamics simulations, we show that the model lipid bilayer can accommodate a higher concentration of triolein (TO) than earlier anticipated, by sequestering triolein molecules to the bilayer center in the form of a disordered, isotropic, mobile neutral lipid aggregate, at least 17 nm in diameter, which forms spontaneously, and remains stable on at least the microsecond time scale. The results give credence to the hotly debated existence of mobile neutral lipid aggregates of unknown function present in malignant cells, and to the early biogenesis of lipid droplets accommodated between the two leaflets of the endoplasmic reticulum membrane. The TO aggregates give the bilayer a blister-like appearance, and will hinder the formation of multi-lamellar phases in model, and possibly living membranes. The blisters will result in anomalous membrane probe partitioning, which should be accounted for in the interpretation of probe-related measurements.

Phase coexistence in a triolein-phosphatidylcholine system. Implications for lysosomal membrane properties.

The effects of tri- and monoglycerides on phospholipid (POPC) membranes were studied using spectroscopical methods. Triolein was found to form two types of POPC-rich membranes, both with POPC or as a three-component system with monopalmitin. These two membrane types were determined as co-existing phases based on their spontaneous and stable separation and named heavy and light phase according to their sedimentation behaviour. Marked differences were seen in the physical properties of these phases, even though only minor compositional variation was detected. The light, less polar phase was found to be less ordered and more fluid and seemed to allow significantly lower amount of water penetration into the membrane-water interface than pure POPC membrane. The heavy phase, apart from their slightly altered water penetration, resembled more a pure POPC membrane. As triglycerides are present in lysosomal membranes, the present results can be seen as an implication for polarity-based water permeability barrier possibly contributing to the integrity of lysosomes.