Atomic force microscopy measurements reveal multiple bonds between Helicobacter pylori blood group antigen binding adhesin and Lewis b ligand.

The strength of binding between the Helicobacter pylori blood group antigen-binding adhesin (BabA) and its cognate glycan receptor, the Lewis b blood group antigen (Le(b)), was measured by means of atomic force microscopy. High-resolution measurements of rupture forces between single receptor-ligand pairs were performed between the purified BabA and immobilized Le(b) structures on self-assembled monolayers. Dynamic force spectroscopy revealed two similar but statistically different bond populations. These findings suggest that the BabA may form different adhesive attachments to the gastric mucosa in ways that enhance the efficiency and stability of bacterial adhesion.

Cadherin adhesion and mechanotransduction.

Cadherins are the principal adhesion proteins at intercellular junctions and function as the biochemical Velcro that binds cells together. Besides this mechanical function, cadherin complexes are also mechanotransducers that sense changes in tension and trigger adaptive reinforcement of intercellular junctions. The assembly and regulation of cadherin adhesions are central to their mechanical functions, and new evidence is presented for a comprehensive model of cadherin adhesion, which is surprisingly more complex than previously appreciated. Recent findings also shed new light on mechanisms that regulate cadherin junction assembly, adhesion, and mechanotransduction. We further describe recent evidence for cadherin-based mechanotransduction, and the rudiments of the molecular mechanism, which involves α-catenin and vinculin as key elements. Potential roles of a broader cast of possible force-sensitive partners are considered, as well as known and speculative biological consequences of adhesion and force transduction at cadherin-mediated junctions.

Bioengineered surfaces promote specific protein-glycan mediated binding of the gastric pathogen Helicobacter pylori.

Helicobacter pylori colonizes the gastric mucosa of half of the worlds population and persistent infection is related with an increase in the risk of gastric cancer. Adhesion of H. pylori to the gastric epithelium, which is essential for infection, is mediated by bacterial adhesin proteins that recognize specific glycan structures (Gly-R) expressed in the gastric mucosa. The blood group antigen binding adhesin (BabA) recognizes difucosylated antigens such as Lewis B (Leb), while the sialic acid binding adhesin (SabA) recognizes sialylated glycoproteins and glycolipids, such as sialyl-Lewis x (sLex). This work aimed to investigate whether these Gly-Rs (Leb and sLex) can attract and specifically bind H. pylori after immobilization on synthetic surfaces (self-assembled monolayers (SAMs) of alkanethiols on gold). Functional bacterial adhesion assays for (Gly-R)-SAMs were performed using H. pylori strains with different adhesin protein profiles. The results demonstrate that H. pylori binding to surfaces occurs via interaction between its adhesins and cognate (Gly-R)-SAMs and bound H. pylori maintains its characteristic rod-shaped morphology only during conditions of specific adhesin-glycan binding. These results offer new insights into innovative strategies against H. pylori infection based on the scavenging of bacteria from the stomach using specific H. pylori chelating biomaterials.

Poly(N-isopropyl acrylamide) brush topography: dependence on grafting conditions and temperature.

The topography of poly (N-isopropyl acrylamide) brushes end-grafted from initiator-terminated monolayers was imaged by atomic force microscopy, as a function of the area per chain and of solvent quality. Measurements were done in air and in water, below and above the lower critical solution temperature. At low grafting densities and molecular weights, area-averaged ellipsometry measurements did not detect changes in the volume of water-swollen, end-grafted polymer films above the lower critical solution temperature. However, atomic force microscopy images revealed surface features that suggest the formation of lateral aggregates or "octopus micelles". At high grafting densities and molecular weights, the films collapsed uniformly, as detected by both AFM imaging and ellipsometry. These findings reconcile in part prior results suggesting that some poly(N-isopropyl acrylamide) chains do not collapse in poor solvent, and they also reveal more complex collapse behavior above the lower critical solution temperature than is commonly assumed. This behavior would influence the ability to tune the functional properties of poly(N-isopropyl acrylamide) coatings.

End-grafted low-molecular-weight PNIPAM does not collapse above the LCST.

The interfacial properties of end-grafted temperature-responsive poly(N-isopropylacryamide) (PNIPAM) were quantified by direct force measurements both above and below the lower critical solution temperature (LCST) of 32 degrees C. The forces were measured between identical, opposing PNIPAM films and between a PNIPAM film and a lipid membrane. At the grafting densities and molecular weights investigated, the polymer extension did not change significantly above the LCST, and the polymers did not adhere. Below the LCST, the force-distance profiles suggest a vertical phase separation, which results in a diluter outer layer and a dense surface proximal layer. At large separations, the force profiles agree qualitatively with simple polymer theory but deviate at small separations. Importantly, at these low grafting densities and molecular weights, the end-grafted PNIPAM does not collapse above the LCST. This finding has direct implications for triggering liposomal drug release with end-grafted PNIPAM, but it increases the temperature range where these short PNIPAM chains function as steric stabilizers.

Similarities between heterophilic and homophilic cadherin adhesion.

The mechanism that drives the segregation of cells into tissue-specific subpopulations during development is largely attributed to differences in intercellular adhesion. This process requires the cadherin family of calcium-dependent glycoproteins. A widely held view is that protein-level discrimination between different cadherins on cell surfaces drives this sorting process. Despite this postulated molecular selectivity, adhesion selectivity has not been quantitatively verified at the protein level. In this work, molecular force measurements and bead aggregation assays tested whether differences in cadherin bond strengths could account for cell sorting in vivo and in vitro. Studies were conducted with chicken N-cadherin, canine E-cadherin, and Xenopus C-cadherin. Both qualitative bead aggregation and quantitative force measurements show that the cadherins cross-react. Furthermore, heterophilic adhesion is not substantially weaker than homophilic adhesion, and the measured differences in adhesion do not correlate with cell sorting behavior. These results suggest that the basis for cell segregation during morphogenesis does not map exclusively to protein-level differences in cadherin adhesion.

Calcium site mutations in cadherin: impact on adhesion and evidence of cooperativity.

This work describes quantitative force and bead aggregation measurements of the adhesion and binding mechanisms of canine E-cadherin mutants W2A, D134A, D103A, D216A, D325A, and D436A. The W2A mutation affects the formation of the N-terminal strand dimer, and the remaining mutations target calcium binding sites at the interdomain junctions. Surface force measurements show that the full ectodomain of canine E-cadherin forms two bound states that span two intermembrane gap distances. The outer bond coincides with adhesion between the N-terminal extracellular domains (EC1) and the inner bond corresponds to adhesion via extracellular domain 3 (EC3). The W2A, D103A, D134A, and D216A mutations all eliminated adhesion between the N-terminal domains, and they attenuated or nearly eliminated the inner bond. The W2A mutant, which does not destabilize the protein structure, attenuates binding via EC3, which is separated from the mutation by several hundred amino acids. This long-range effect suggests that the presence or absence of tryptophan-2 docking allosterically alters the adhesive function of distal sites on the protein. This finding appears to reconcile the multidomain binding mechanism with mutagenesis studies, which suggested that W2 is the sole binding interface. The effects of the calcium site mutations indicate that structural perturbations cooperatively impact large regions of the protein structure. However, the influence of the calcium sites on cadherin structure and function depends on their location in the protein.

Multiple-bond kinetics from single-molecule pulling experiments: evidence for multiple NCAM bonds.

The kinetic parameters of single bonds between neural cell adhesion molecules were determined from atomic force microscope measurements of the forced dissociation of the homophilic protein-protein bonds. The analytical approach described provides a systematic procedure for obtaining rupture kinetics for single protein bonds from bond breakage frequency distributions obtained from single-molecule pulling experiments. For these studies, we used the neural cell adhesion molecule (NCAM), which was recently shown to form two independent protein bonds. The analysis of the bond rupture data at different loading rates, using the single-bond full microscopic model, indicates that the breakage frequency distribution is most sensitive to the distance to the transition state and least sensitive to the molecular spring constant. The analysis of bond failure data, however, motivates the use of a double-bond microscopic model that requires an additional kinetic parameter. This double-bond microscopic model assumes two independent NCAM-NCAM bonds, and more accurately describes the breakage frequency distribution, particularly at high loading rates. This finding agrees with recent surface-force measurements, which showed that NCAM forms two spatially distinct bonds between opposed proteins.

pH-dependent mucoadhesion of a poly(N-isopropylacrylamide) copolymer reveals design rules for drug delivery.

This study investigated the mucoadhesive property of a hydrophobically modified copolymer N-isopropylacryamide and glycidylacrylamide NIPAM-N-Gly-(C18)2 (NIPAM-Gly). Prior studies demonstrated that the interfacial properties of this copolymer are pH dependent and that the chains form strong hydrogen bonds at pH < 7 via the carboxylic acid side chains of the glycine moieties. Mucin interactions with the copolymer brushes were investigated by surface plasmon resonance and by direct force measurements. Mucin adsorption was determined as a function of pH, ionic strength, and mucin concentration. It adsorbs to the copolymer strongly at pH 5, but the adsorption decreases with increasing pH. The adsorbed amount is also ionic-strength dependent, decreasing with increasing monovalent salt concentrations at all pH values investigated. When compared with similar investigations with poly(ethylene oxide), these results provide insights into both the chemical characteristics and the solution conditions that determine the mucoadhesive properties of polymers.

Mechanism of homophilic adhesion by the neural cell adhesion molecule: use of multiple domains and flexibility.

The extracellular regions of adhesion proteins of the Ig superfamily comprise multiple, tandemly arranged domains. We used directforce measurements to investigate how this modular architecture contributes to the adhesive interactions of the neural cell adhesion molecule (NCAM), a representative of this protein class. The extracellular region of NCAM comprises five immunoglobulin and two fibronectin domains. Previous investigations generated different models for the mechanism of homophilic adhesion that each use different domains. We use force measurements to demonstrate that NCAM binds in two spatially distinct configurations. Igdomain deletion mutants identified the domains responsible for each of the adhesive bonds. The measurements also confirmed the existence of a flexible hinge that alters the orientation of the adhesive complexes and the intermembrane distance. These results suggest that a combination of multiple bound states and internal molecular flexibility allows for sequentially synergistic bond formation and the ability to accommodate differences in intercellular space.

Forced dissociation of the strand dimer interface between C-cadherin ectodomains.

The force-induced dissociation of the strand dimer interface in C-cadherin has been studied using steered molecular dynamics simulations. The dissociation occurred, without domain unraveling, after the extraction of the conserved trypthophans (Trp2) from their respective hydrophobic pockets. The simulations revealed two stable positions for the Trp2 side chain inside the pocket. The most internal stable position involved a hydrogen bond between the ring Nepsilon of Trp2 and the backbone carbonyl of Glu90. In the second stable position, the aromatic ring is located at the pocket entrance. After extracting the two tryptophans from their pockets, the complex exists in an intermediate bound state that involves a close packing of the tryptophans with residues Asp1 and Asp27 from both domains. Dissociation occurred after this residue association was broken. Simulations carried out with a complex formed between W2A mutants showed that the mutant complex dissociates more easily than the wild type complex does. These results correlate closely with the role of the conserved tryptophans suggested previously by site directed mutagenesis.

Tuning the interfacial properties of grafted chains with a pH switch.

Environmentally responsive, water-soluble polymers have a wide variety of uses ranging from drug delivery to viscosity modifiers. Their utility lies in the ability to use environmental perturbations to dramatically alter the material properties. Here, we describe the interfacial properties of a hydrophobically modified copolymer of N-isopropylacrylamide and glycinylacrylamide (NIPAM-N-Gly-(C18)2), which is both temperature and pH responsive. Direct force measurements quantified the substantial pH-dependent change in the molecular properties of end-grafted NIPAM-N-Gly-(C18)2 monolayers. At pH 8.0, where the glycine side chains are ionized, the polymers exhibit stereotypical polyelectrolyte behavior. Side chain neutralization at pH 5.0 causes a substantial decrease in the film thickness, and the polymer films adhere strongly. The adhesion is presumably through H-bonding between the glycine side chains. Our findings revealed the likely molecular basis of pH-dependent changes in the copolymer films and identified clear design criteria for tuning the interfacial properties of these polymer films.

Functional analysis of the structural basis of homophilic cadherin adhesion.

The structures of many cell surface adhesion proteins comprise multiple tandem repeats of structurally similar domains. In many cases, the functional significance of this architecture is unknown, and there are several cases in which evidence for individual domain involvement in adhesion has been contradictory. In particular, the extracellular region of the adhesion glycoprotein cadherin consists of five tandemly arranged domains. One proposed mechanism postulated that adhesion involves only trans interactions between the outermost domains. However, subsequent investigations have generated several competing models. Here we describe direct measurements of the distance-dependent interaction potentials between cadherin mutants lacking different domains. By quantifying both the absolute distances at which opposed cadherin fragments bind and the quantized changes in the interaction potentials that result from deletions of individual domains, we demonstrate that two domains participate in homophilic cadherin binding. This finding contrasts with the current view that cadherins bind via a single, unique site on the protein surface. The potentials that result from interactions involving multiple domains generate a novel, modular binding mechanism in which opposed cadherin ectodomains can adhere in any of three antiparallel alignments.

Forced detachment of the CD2-CD58 complex.

The force-induced detachment of the adhesion protein complex CD2-CD58 was studied by steered molecular dynamics simulations. The forced detachment of CD2 and CD58 shows that the system can respond to an external force by two mechanisms, which depend on the loading rate. At the rapid loading rates of 70 and 35 pN/ps (pulling speeds of 1 and 0.5 A/ps) the two proteins unfold before they separate, whereas at slower loading rates of 7 and 3.5 pN/ps (pulling speeds of 0.1 and 0.05 A/ps), the proteins separate before the domains can unfold. When subjected to a constant force of 400 pN, the two proteins separated without significant structural distortion. These findings suggest that protein unfolding is not coupled to the adhesive function of CD2 and CD58. The simulations further confirm that salt bridges primarily determine the tensile strength of the protein-to-protein bond, and that the order of salt bridge rupture depends mainly on the position of the bond, relative to the line of action of the applied force. Salt bridges close to this line break first. The importance of each of the salt bridges for adhesion, determined from the simulations, correlates closely with their role in cell-to-cell adhesion and equilibrium binding determined by site-directed mutagenesis experiments.

Force as a probe of membrane protein structure and function.

Force measurement techniques are being used increasingly to explore the mechanical properties of proteins, as well as the structural origins of intermolecular forces. Developments in instrumentation and the increasing availability of engineered and purified membrane proteins have widely expanded the range of biological systems that can be addressed. Within the past year, force measurements have identified novel mechanisms of binding between cell-surface proteins, as well as the mechanical properties of integral membrane proteins and the intramolecular interactions that stabilize their structures.

Chain-length dependence of the protein and cell resistance of oligo(ethylene glycol)-terminated self-assembled monolayers on gold.

Oligo(ethylene glycol) (O-EG(n))-terminated alkanethiol surface-assembled monolayers (SAMs) have been reported to demonstrate protein-resistant properties similar to those of poly(ethylene glycol) (PEG). In this study, we compared the relative protein resistance of short and long ethylene oxide chains, SAMs of PEG 5000, PEG 2000, O-EG(3) (molecular weight = 120), and O-EG(6) (molecular weight = 240), on gold surfaces. Surface plasmon resonance showed that these monolayers were all protein-resistant within the uncertainty of the measurement. However, they exhibited different adhesive properties toward 3T3 mouse fibroblast adhesion in supplemented Dulbecco's modified Eagles medium. The results show that the cell adhesion was sensitive to the concentration of proteins supplemented in the culture medium and to the length of PEG chains.

Long-term stability of grafted polyethylene glycol surfaces for use with microstamped substrates in neuronal cell culture.

Crucial to long-term stability of neuronal micropatterns is functional retention of the underlying substratum while exposed to cell culture conditions. We report on the ability of covalently bound PEG films in long-term cell culture to continually retard protein adhesion and cell growth. PDMS microstamps were used to create poly-d-lysine (PDL) substrates permissive to cell attachment and growth, and polyethylene glycol (PEG) substrates were used to minimize protein and cell adhesion. Film thickness was measured using null ellipsometry and atomic force microscopy (AFM). Organosilane film structure was examined using Fourier transform infrared (FT-IR) spectroscopy. Long-term film stability in cell culture conditions was tested by immersion in 0.1 M sodium phosphate buffer pH 7.4 for up to one month. Null ellipsometry and water contact measurements indicated that organosilane films were stable up to one month, whereas the PEG film thickness declined rapidly after day 25. Hippocampal cells plated at 200 cells/mm2 on uniform PEG substrates gave a steady increase in biofilm thickness on PEG films throughout the culture, possibly from proteins of neuronal origin. We found that all the layers in the cross-linking procedure were stable in cell culture conditions, with the exception of PEG, which degraded after day 25.

Direct measurements of multiple adhesive alignments and unbinding trajectories between cadherin extracellular domains.

Direct measurements of the interactions between antiparallel, oriented monolayers of the complete extracellular region of C-cadherin demonstrate that, rather than binding in a single unique orientation, the cadherins adhere in three distinct alignments. The strongest adhesion is observed when the opposing extracellular fragments are completely interdigitated. A second adhesive alignment forms when the interdigitated proteins separate by 70 +/- 10 A. A third complex forms at a bilayer separation commensurate with the approximate overlap of cadherin extracellular domains 1 and 2 (CEC1-2). The locations of the energy minima are independent of both the surface density of bound cadherin and the stiffness of the force transducer. Using surface element integration, we show that two flat surfaces that interact through an oscillatory potential will exhibit discrete minima at the same locations in the force profile measured between hemicylinders covered with identical materials. The measured interaction profiles, therefore, reflect the relative separations at which the antiparallel proteins adhere, and are unaffected by the curvature of the underlying substrate. The successive formation and rupture of multiple protein contacts during detachment can explain the observed sluggish unbinding of cadherin monolayers. Velocity-distance profiles, obtained by quantitative video analysis of the unbinding trajectory, exhibit three velocity regimes, the transitions between which coincide with the positions of the adhesive minima. These findings suggest that cadherins undergo multiple stage unbinding, which may function to impede adhesive failure under force.

A quantitative assessment of heterogeneity for surface-immobilized proteins.

Many biotechnological applications use protein receptors immobilized on solid supports. Although, in solution, these receptors display homogeneous binding affinities and association/dissociation kinetics for their complementary ligand, they often display heterogeneous binding characteristics after immobilization. In this study, a fluorescence-based fiber-optic biosensor was used to quantify the heterogeneity associated with the binding of a soluble analyte, fluorescently labeled trinitrobenzene, to surface-immobilized monoclonal anti-TNT antibodies. The antibodies were immobilized on silica fiber-optic probes via five different immobilization strategies. We used the Sips isotherm to assesses and compare the heterogeneity in the antibody binding affinity and kinetic rate parameters for these different immobilization schemes. In addition, we globally analyzed kinetic data with a two-compartment transport-kinetic model to analyze the heterogeneity in the analyte-antibody kinetics. These analyses provide a quantitative tool by which to evaluate the relative homogeneity of different antibody preparations. Our results demonstrate that the more homogeneous protein preparations exhibit more uniform affinities and kinetic constants.