Influence of self-assembled monolayer surface chemistry on Candida antarctica lipase B adsorption and specific activity

Immobilization of Candida antarctica B lipase was examined on gold surfaces modified with either methyl- or hydroxyl-terminated self-assembled alkylthiol monolayers (SAMs), representing hydrophobic and hydrophilic surfaces, respectively. Lipase adsorption was monitored gravimetrically using a quartz crystal microbalance. Lipase activity was determined colorimetrically by following p-nitrophenol propionate hydrolysis. Adsorbed lipase topography was examined by atomic force microscopy. The extent of lipase adsorption was nearly identical on either surface (approximately 240 ng cm⁻²), but its specific activity was sixfold higher on the methyl-terminated SAM, showing no activity loss upon immobilization. A uniform, 5.5 nm high, highly packed monolayer of CALB formed on the methyl-terminated SAM, while the adsorbed protein was disordered on the hydroxyl-terminated SAM. Hydrophobic surfaces thus may specifically orient the lipase in a highly active state.     

Molecular dynamics simulations of a protein on hydrophobic and hydrophilic surfaces.

Molecular dynamics simulations have been used to investigate the behavior of the peripheral membrane protein, cytochrome c, covalently tethered to hydrophobic (methyl-terminated) and hydrophilic (thiol-terminated) self-assembled monolayers (SAMs). The simulations predict that the protein will undergo minor structural changes when it is tethered to either surface, and the structures differ qualitatively on the two surfaces: the protein is less spherical on the hydrophilic SAM where the polar surface residues reach out to interact with the SAM surface. The protein is completely excluded from the hydrophobic SAM but partially dissolves in the hydrophilic SAM. Consequently, the surface of the thiol-terminated SAM is considerably less ordered than that of the methyl-terminated SAM, although a comparable, high degree of order is maintained in the bulk of both SAMs: the chains exhibit collective tilts in the nearest-neighbor direction at angles of 20 degrees and 17 degrees with respect to the surface normal in the hydrophobic and the hydrophilic SAMs, respectively. On the hydrophobic SAM the protein is oriented so that the heme plane is more nearly parallel to the surface, whereas on the hydrophilic surface it is more nearly perpendicular. The secondary structure of the protein, dominated by alpha helices, is not significantly affected, but the structure of the loops as well as the helix packing is slightly modified by the surfaces.     

Bacterial adhesion to hydroxyl- and methyl-terminated alkanethiol self-assembled monolayers.

The attachment of bacteria to solid surfaces is influenced by substratum chemistry, but to determine the mechanistic basis of this relationship, homogeneous, well-defined substrata are required. Self-assembled monolayers (SAMs) were constructed from alkanethiols to produce a range of substrata with different exposed functional groups, i.e., methyl and hydroxyl groups and a series of mixtures of the two. Percentages of hydroxyl groups in the SAMs and substratum wettability were measured by X-ray photoelectron spectroscopy and contact angles of water and hexadecane, respectively. SAMs exhibited various substratum compositions and wettabilities, ranging from hydrophilic, hydroxyl-terminated monolayers to hydrophobic, methyl-terminated monolayers. The kinetics of attachment of an estuarine bacterium to these surfaces in a laminar flow chamber were measured over periods of 120 min. The initial rate of net adhesion, the number of cells attached after 120 min, the percentage of attached cells that adsorbed or desorbed between successive measurements, and the residence times of attached cells were quantified by phase-contrast microscopy and digital image processing. The greatest numbers of attached cells occurred on hydrophobic surfaces, because (i) the initial rates of adhesion and the mean numbers of cells that attached after 120 min increased with the methyl content of the SAM and the contact angle of water and (ii) the percentage of cells that desorbed between successive measurements (ca. 2 min) decreased with increasing substratum hydrophobicity. With all surfaces, 60 to 80% of the cells that desorbed during the 120-min exposure period had residence times of less than 10 min, suggesting that establishment of firm adhesion occurred quickly on all of the test surfaces.     

Adsorption of zein on surfaces with controlled wettability and thermal stability of adsorbed zein films

Adsorption characteristics of zein protein on hydrophobic and hydrophilic surfaces have been investigated to understand the orientation changes associated with the protein structure on a surface. The protein is adsorbed by a self-assembly procedure on a monolayer-modified gold surface. It is observed that zein shows higher affinity toward hydrophilic than hydrophobic surfaces on the basis of the initial adsorption rate followed by quartz crystal microbalance studies. Reflection absorption infrared (RAIR) spectroscopic studies reveal the orientation changes associated with the adsorbed zein films. Upon adsorption, the protein is found to be denatured and the transformation of alpha-helix to beta-sheet form is inferred. This transformation is pronounced when the protein is adsorbed on hydrophobic surfaces as compared to hydrophilic surfaces. Electrochemical techniques (cyclic voltammetry and impedance techniques) are very useful in assessing the permeability of zein film. It is observed that the zein moieties adsorbed on hydrophilic surfaces are highly impermeable in nature and act as a barrier for small molecules. The topographical features of the deposits before and after adsorption are analyzed by atomic force microscopy. The protein adsorbed on hydrophilic surface shows rod- and disclike features that are likely to be the base units for the growth of cylindrical structures of zein. The thermal stability of the adsorbed zein film has been followed by variable-temperature RAIR measurements.     

Adsorption Properties and Activities of Lipase on a Gold Substrate Modified by Self-assembled Monolayers

The adsorption properties, amount and specific activity of lipase D from Rhizopus delemar were investigated by employing a gold substrate modified with seven kinds of thiol monolayer. Quartz crystal microbalance measurements revealed that the amount of the enzyme adsorbed to the hydrophobic monolayers (e.g. benzenethiol) was much higher than that to the hydrophilic monolayers (e.g. 3-mercaptopropanoic acid). In contrast, lipase D adsorbed to the hydrophilic, 2-amino-1-ethanethiol monolayer showed the highest specific activity, the value being 300-fold higher than for the same enzyme dissolved in an aqueous medium.     

Adsorption properties and activities of lipase on a gold substrate modified by self-assembled monolayers.

The adsorption properties, amount and specific activity of lipase D from Rhizopus delemar were investigated by employing a gold substrate modified with seven kinds of thiol monolayer. Quartz crystal microbalance measurements revealed that the amount of the enzyme adsorbed to the hydrophobic monolayers (e.g. benzenethiol) was much higher than that to the hydrophilic monolayers (e.g. 3-mercaptopropanoic acid). In contrast, lipase D adsorbed to the hydrophilic, 2-amino-1-ethanethiol monolayer showed the highest specific activity, the value being 300-fold higher than for the same enzyme dissolved in an aqueous medium.     

Adsorption, lubrication, and wear of lubricin on model surfaces: polymer brush-like behavior of a glycoprotein

Using a surface force apparatus, we have measured the normal and friction forces between layers of the human glycoprotein lubricin, the major boundary lubricant in articular joints, adsorbed from buffered saline solution on various hydrophilic and hydrophobic surfaces: i), negatively charged mica, ii), positively charged poly-lysine and aminothiol, and iii), hydrophobic alkanethiol monolayers. On all these surfaces lubricin forms dense adsorbed layers of thickness 60–100 nm. The normal force between two surfaces is always repulsive and resembles the steric entropic force measured between layers of end-grafted polymer brushes. This is the microscopic mechanism behind the antiadhesive properties showed by lubricin in clinical tests. For pressures up to ~6 atm, lubricin lubricates hydrophilic surfaces, in particular negatively charged mica (friction coefficient μ = 0.02–0.04), much better than hydrophobic surfaces (μ > 0.3). At higher pressures, the friction coefficient is higher (μ > 0.2) for all surfaces considered and the lubricin layers rearrange under shear. However, the glycoprotein still protects the underlying substrate from damage up to much higher pressures. These results support recent suggestions that boundary lubrication and wear protection in articular joints are due to the presence of a biological polyelectrolyte on the cartilage surfaces.     

Cationic carbosilane dendrimers-lipid membrane interactions

The aim of this work was to study interactions between cationic carbosilane dendrimers (CBS) and lipid bilayers or monolayers. Two kinds of second generation carbosilane dendrimers were used: NN16 with Si-O bonds and BDBR0011 with Si-C bonds. The results show that cationic carbosilane dendrimers interact both with liposomes and lipid monolayers. Interactions were stronger for negatively charged membranes and high concentration of dendrimers. In liposomes interactions were studied by measuring fluorescence anisotropy changes of fluorescent labels incorporated into the bilayer. An increase in fluorescence anisotropy was observed for both fluorescent probes when dendrimers were added to lipids that means the decreased membrane fluidity. Both the hydrophobic and hydrophilic parts of liposome bilayers became more rigid. This may be due to dendrimers' incorporation into liposome bilayer. For higher concentrations of both dendrimers precipitation occurred in negatively charged liposomes. NN16 dendrimer interacted stronger with hydrophilic part of bilayers whereas BDBR0011 greatly modified the hydrophobic area. Monolayers method brought similar results. Both dendrimers influenced lipid monolayers and changed surface pressure. For negatively charged lipids the monitored parameter changed stronger than for uncharged DMPC lipids. Moreover, NN16 dendrimer interacted stronger than the BDBR0011.     

Graphite-like lubrication of mesothelium by oligolamellar pleural surfactant.

Six studies have been completed to reevaluate pleural surfactant as a possible boundary lubricant in mesothelial sliding. It is capable of remarkable antiwear action, giving a mean scar diameter on a standard "four-ball test" comparable to the best commercially available lubricants and reducing friction to values anticipated from lamellated solid lubricants such as graphite. Pleural surfaces displayed appreciable hydrophobicity, which was almost eliminated by rinsing with a lipid solvent from which phospholipid was recovered and quantified. These quantities indicated that equivalent of 7.3 adsorbed monolayers of surface-active phospholipid, which was in general agreement with the number of layers of a graphite-like surface coating visualized by electron microscopy by use of a novel fixation procedure that avoids conventional aldehydes known to destroy hydrophobic surfaces. Graphite-like (dry) lubrication by adsorbed surface-active phospholipid is discussed as an excellent lubrication system available wherever the distribution of fluid allows the pleura to make contact.     

Effects of lithium on the human erythrocyte membrane and molecular models

The mechanism whereby lithium carbonate controls manic episodes and possibly influences affective disorders is not yet known. There is evidence, however, that lithium alters sodium transport and may interfere with ion exchange mechanisms and nerve conduction. For these reasons it was thought of interest to study its perturbing effects upon membrane structures. The effects of lithium carbonate (Li+) on the human erythrocyte membrane and molecular models have been investigated. The molecular models consisted in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representing classes of phospholipids located in the outer and inner monolayers of the erythrocyte membrane, respectively. This report presents the following evidence that Li+ interacts with cell membranes: a) X-ray diffraction indicated that Li+ induced structural perturbation of the polar head group and of the hydrophobic acyl regions of DMPC and DMPE; b) experiments performed on DMPC large unilamellar vesicles (LUV) by fluorescence spectroscopy also showed that Li+ interacted with the lipid polar groups and hydrophobic acyl chains, and c) in scanning electron microscopy (SEM) studies on intact human erythrocytes the formation of echinocytes was observed, effect that might be due to the insertion of Li+ in the outer monolayer of the red cell membrane.     

Effects of the local anesthetic benzocaine on the human erythrocyte membrane and molecular models

The interaction of the local anesthetic benzocaine with the human erythrocyte membrane and molecular models is described. The latter consisted of isolated unsealed human erythrocyte membranes (IUM), large unilamellar vesicles (LUV) of dimyristoylphospatidylcholine (DMPC), and phospholipid multilayers of DMPC and dimyristoylphospatidyletanolamine (DMPE), representatives of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Optical and scanning electron microscopy of human erythrocytes revealed that benzocaine induced the formation of echinocytes. Experiments performed on IUM and DMPC LUV by fluorescence spectroscopy showed that benzocaine interacted with the phospholipid bilayer polar groups and hydrophobic acyl chains. X-ray diffraction analysis of DMPC confirmed these results and showed that benzocaine had no effects on DMPE. The effect on sodium transport was also studied using the isolated toad skin. Electrophysiological measurements indicated a significant decrease in the potential difference (PD) and in the short-circuit current (Isc) after the application of benzocaine, reflecting inhibition of active ion transport.     

Orientation of anthracyclines in lipid monolayers and planar asymmetrical bilayers: a surface-enhanced resonance Raman scattering study.

The interaction of anthracyclines (daunorubicin and idarubicin) with monolayers of zwitterionic palmitoyloleoylphosphatidylcholine (POPC) and anionic dipalmitoylphosphatidic acid (POPC-DPPA 80-20 mol%) was studied by surface pressure measurements and compared with previous results obtained with other anthracyclines (pirarubicin and adriamycin). These anthracycline/phospholipid monolayers were next transferred by a Langmuir-Blodgett technique onto planar supports and studied by surface-enhanced resonance Raman scattering (SERRS), which gave information about the orientation of anthracycline in the monolayers. On the whole, the adsorption of anthracyclines in zwitterionic monolayers increases with the anthracycline hydrophobic/hydrophilic balance, which underlines the role of the hydrophobic component of the interaction. On the contrary, the anthracyclines remain adsorbed on the polar headgroups of the phospholipids in the presence of DPPA and form a screen that limits a deeper penetration of other anthracycline molecules. To study by SERRS measurements the crossing of pirarubicin through a phospholipid bilayer used as a membrane model, asymmetrical POPC-DPPA/POPC or POPC/POPC-DPPA bilayers were transferred by the Langmuir-Schäfer method, thanks to a laboratory-built set-up, and put in contact with a pirarubicin aqueous solution. It has been shown that the presence of anionic DPPA in the first monolayer in contact with pirarubicin would limit its crossing. This limiting effet is not observed if the first monolayer is zwitterionic.     

Lipid monolayers on Hg as a valid experimental model for lipid membranes under electrical fields

Monolayers spread on Hg drops are shown as a suitable experimental set up to study the influence of external electric fields on the structure of lipid membranes. The electrical response exhibits a sharp transition at 24 °C, the transition temperature of DMPC. In addition, voltammetric response of monolayers of mixtures of DMPC/DMPE adsorbed on mercury, shows a similar trend to that found for dipole potential of monolayers of the same composition spread on an air-solution interface.It is concluded that a lipid monolayer adsorbed in a mercury-solution interface, has comparable properties as those found in other experimental models of lipid membranes in similar conditions. In addition, they constitute an ideal set up to study the effect of electrical fields on the dynamic conformation of lipids as a function of packing change produced by the condensation in the gel state or by the interaction of polar head groups.     

QCM-D sensitivity to protein adsorption reversibility

Using a quartz crystal microbalance with dissipative monitoring (QCM-D) we have determined the adsorption reversibility and viscoelastic properties of ribonuclease A adsorbed to hydrophobic self-assembled monolayers. Consistent with previous work with proteins unfolding on hydrophobic surfaces, high protein solution concentrations, reduced adsorption times, and low ammonium sulfate concentrations lead to increased adsorption reversibility. Measured rigidity of the protein layers normalized for adsorbed protein amounts, a quantity we term specific dissipation, correlated with adsorption reversibility of ribonuclease A. These results suggest that specific dissipation may be correlated with changes in structure of adsorbed proteins.     

Interactions of thrombin with fibrinogen adsorbed on methyl-, hydroxyl-, amine-, and carboxyl-terminated self-assembled monolayers

We have examined the initial phase of fibrin formation, thrombin-catalyzed fibrinopeptide cleavage, from adsorbed fibrinogen using surface plasmon resonance and liquid chromatography-mass spectrometry. Fibrinogen adsorption impaired thrombin-fibrinogen interactions compared to the interactions of thrombin with fibrinogen in solution. The properties of the underlying substrate significantly affected the extent and kinetics of fibrinopeptide cleavage, and the conversion of adsorbed fibrinogen to fibrin. Fibrinogen adsorbed on negatively charged surfaces (carboxyl-terminated self-assembled monolayers) released a smaller amount of fibrinopeptides, at a reduced rate relative to those of hydrophobic, hydrophilic, and positively charged surfaces (methyl-, hydroxyl-, and amine-terminated self-assembled monolayers, respectively). Additionally, the conversion of adsorbed fibrinogen to fibrin was comparatively inefficient at the negatively charged surface. These data correlated well with trends previously reported for fibrin proliferation as a function of surface properties. We conclude that thrombin interactions with adsorbed fibrinogen determine the extent of subsequent fibrin proliferation on surfaces.     

Nisin adsorption on hydrophilic and hydrophobic surfaces: evidence of its interactions and antibacterial activity

Study of peptides adsorption on surfaces remains a current challenge in literature. A complementary approach, combining X‐ray photoelectron spectroscopy (XPS) and time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) was used to investigate the antimicrobial peptide nisin adsorption on hydrophilic and hydrophobic surfaces. The native low density polyethylene was used as hydrophobic support and it was grafted with acrylic acid to render it hydrophilic. XPS permitted to confirm nisin adsorption and to determine its amount on the surfaces. ToF‐SIMS permitted to identify the adsorbed bacteriocin type and to observe its distribution and orientation behavior on both types of surfaces. Nisin was more oriented by its hydrophobic side to the hydrophobic substrate and by its hydrophilic side to the outer layers of the adsorbed peptide, in contrast to what was observed on the hydrophilic substrate. A correlation was found between XPS and ToF‐SIMS results, the types of interactions on both surfaces and the observed antibacterial activity. Such interfacial studies are crucial for better understanding the peptides interactions and adsorption on surfaces and must be considered when setting up antimicrobial surfaces. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Interactions between adsorbed layers of alphaS1-casein with covalently bound side chains: a self-consistent field study

The effect on the adsorbed layer properties of the modification of alpha S1-casein by covalent bonding with an uncharged polysaccharide side chain has been investigated using lattice-based self-consistent field (SCF) theory. Interactions between two hydrophobic planar surfaces coated by a layer of adsorbed modified alpha S1-casein have been studied as a function of pH and ionic strength. While the interactions of the unmodified alpha S1-casein layers become attractive at high ionic strength, it has been shown that the presence of polysaccharide attachment to the alpha S1-casein molecule can confer net repulsive interactions over a wide range of salt concentration. The disordered protein is represented as a linear flexible polyampholyte with a sequence of hydrophobic, polar, and charged units based on the known alpha S1-casein primary structure. The hydrophilic side chain is attached at various fixed positions along the casein backbone. Different lengths and locations of the attached polysaccharide side chain are examined. Interfacial structures and colloidal stability properties of the system are determined, including the surface-surface interaction potential, the extent of protein bridging, and the distribution of protein segments from the surface under different conditions of pH and ionic strength. It has been found that the covalent bonding of short hydrophilic chains may not only enhance but can also worsen the colloidal stabilizing properties of the modified protein, depending on the position of the attachment.     

A generic expression system to produce proteins that co-assemble with alkane thiol SAM

Surface biology aims to observe and control biological processes by combining bio-, surface, and physical chemistry. Self-assembled monolayers (SAM) on gold surfaces have provided excellent methods for nanoscale surface preparation for such studies. However, extension of this work requires the specific immobilization of whole protein domains and the direct incorporation of recombinant proteins into SAM is still problematic. In this study a short random coil peptide has been designed to insert into thioalkane layers by formation of a hydrophobic helix. Surface plasmon resonance (SPR) studies show that specific immobilization via the internal cysteine is achieved. Addition of the peptide sequence to the terminus of a protein at the genetic level enables the production of a range of recombinant fusion-proteins with good yield. SPR shows that the proteins display the same gold-binding behavior as the peptide. It is shown that cell growth control can be achieved by printing the proteins using soft lithography with subsequent infilling with thio-alkanes The expression plasmid is constructed so that any stable protein domain can be easily cloned, expressed, purified and immobilized.     

Backbone and sidechain 1H, 13C and 15N chemical shift assignments of the hydrophobin DewA from Aspergillus nidulans

Hydrophobins are proteins secreted by filamentous fungi that are able to self-assemble into monolayers at hydrophobic:hydrophilic interfaces. The layers are amphipathic and can reverse the wettability of surfaces. Hydrophobins have several roles in fungal development, including the formation of coatings on fungal structures to render them hydrophobic. Here we report the backbone and sidechain assignments for the class I hydrophobin DewA from the fungus Aspergillus nidulans.