Probing multivalency in ligand-receptor-mediated adhesion of soft, biomimetic interfaces.

Many biological functions at cell level are mediated by the glycocalyx, a dense carbohydrate-presenting layer. In this layer specific interactions between carbohydrate ligands and protein receptors are formed to control cell-cell recognition, cell adhesion and related processes. The aim of this work is to shed light on the principles of complex formation between surface anchored carbohydrates and receptor surfaces by measuring the specific adhesion between surface bound mannose on a concanavalin A (ConA) layer via poly(ethylene glycol)-(PEG)-based soft colloidal probes (SCPs). Special emphasis is on the dependence of multivalent presentation and density of carbohydrate units on specific adhesion. Consequently, we first present a synthetic strategy that allows for controlled density variation of functional groups on the PEG scaffold using unsaturated carboxylic acids (crotonic acid, acrylic acid, methacrylic acid) as grafting units for mannose conjugation. We showed by a range of analytic techniques (ATR-FTIR, Raman microscopy, zeta potential and titration) that this synthetic strategy allows for straightforward variation in grafting density and grafting length enabling the controlled presentation of mannose units on the PEG network. Finally we determined the specific adhesion of PEG-network-conjugated mannose units on ConA surfaces as a function of density and grafting type. Remarkably, the results indicated the absence of a molecular-level enhancement of mannose/ConA interaction due to chelate- or subsite-binding. The results seem to support the fact that weak carbohydrate interactions at mechanically flexible interfaces hardly undergo multivalent binding but are simply mediated by the high number of ligand-receptor interactions.

A novel contact model for AFM indentation experiments on soft spherical cell-like particles.

The use of the simple Hertz model for the analysis of Atomic Force Microscopy (AFM) force-distance curves measured on soft spherical cell-like particles leads to significant underestimations of the objects Young's modulus E. To correct this error, a mixed double contact model (based on the simple Hertz model and the Johnson-Kendall-Roberts (JKR) model) was derived. The model considers two independent particle deformation sites: (i) the upper part of the particle is deformed by the AFM indenter, (ii) the bottom part is deformed by the substrate, which is usually unnoticed. It becomes apparent that for soft particles even small forces between substrate and particle can influence the resulting force-distance curves. For instance we show, that a gravity-induced compression on the particle bottom side can have significant influence on the measurements. To highlight these observations, the deviation of the particle Young's modulus E between the simple Hertz model and our model is calculated. This error strongly depends on the ratio of the three involved radii: (i) the radius of the AFM indenter, (ii) the radius of the particle and (iii) the radius of the substrate as well as on the acting gravity force. Overall, the analysis suggests that for nanoscopic indenters the deviation is negligible, whereas the use of microscopic indenters results in significant errors that can be corrected via the presented model. This is important especially for very soft particles, since larger indenters can achieve higher signal to noise ratios. Furthermore, the applicability of the model was confirmed by indentation experiments on hydrogel microbeads. The mixed double contact model is applicable to a large range of indenter geometries and can be adapted for other contact models.

Specific adhesion of carbohydrate hydrogel particles in competition with multivalent inhibitors evaluated by AFM.

Synthetic glycooligomers have emerged as valuable analogues for multivalent glycan structures in nature. These multivalent carbohydrates bind to specific receptors and play a key role in biological processes. In this work, we investigate the specific interaction between mannose ligand presenting soft colloidal probes (SCPs) attached to an atomic force microscope (AFM) cantilever and a Concanavalin A (ConA) receptor surface in the presence of competing glycooligomer ligands. We studied the SCP-ConA adhesion energy via the JKR approach and AFM pull-off experiments in combination with optical microscopy allowing for simultaneous determination of the contact area between SCP and ConA surface. We varied the contact time, loading rate and loading force and measured the resulting mannose/ConA interaction. The average adhesion energy per mannose ligand on the probe was 5 kJ/mol, suggesting that a fraction of mannose ligands presented on the SCP bound to the receptor surface. Adhesion measurements via competitive binding of the SCP in the presence of multivalent glycooligomer ligands did not indicate an influence of their multivalency on the glycooligomer displacement from the ConA surface. The absence of this "multivalency effect" indicates that glycooligomers and ConA do not associate via chelate complexes and shows that steric shielding by the glycooligomers does not slow their displacement upon competitive binding of a ligand presenting surface. These results highlight the high reversibility of carbohydrate-surface interactions, which could be an essential feature of recognition processes on the cell surface.

Metal-mediated molecular self-healing in histidine-rich mussel peptides.

Mussels withstand high-energy wave impacts in rocky seashore habitats by fastening tightly to surfaces with tough and self-healing proteinaceous fibers called byssal threads. Thread mechanical behavior is believed to arise from reversibly breakable metal coordination cross-links embedded in histidine-rich protein domains (HRDs) in the principle load-bearing proteins comprising the fibrous thread core. In order to investigate HRD behavior at the molecular level, we have synthesized a histidine-rich peptide derived from mussel proteins (His5-bys) and studied its reversible adhesive self-interaction in the presence and absence of metal ions using PEG-based soft-colloidal probes (SCPs). Adhesion energies of greater than 0.3 mJ/m(2) were measured in the presence of metal ions, and the stiffness of the modified SCPs exhibited a 3-fold increase, whereas no adhesion was observed in the absence of metals. Raman spectroscopy confirmed the presence of metal-coordination via histidine residues by the peptide-supporting the role of His-metal complexes in the mechanical behavior of the byssus.

Carbohydrate-lectin recognition of sequence-defined heteromultivalent glycooligomers.

Multivalency as a key principle in nature has been successfully adopted for the design and synthesis of artificial glycoligands by attaching multiple copies of monosaccharides to a synthetic scaffold. Besides their potential in various applied areas, e.g. as antiviral drugs, for the vaccine development and as novel biosensors, such glycomimetics also allow for a deeper understanding of the fundamental aspects of multivalent binding of both artificial and natural ligands. However, most glycomimetics so far neglect the purposeful arranged heterogeneity of their natural counterparts, thus limiting more detailed insights into the design and synthesis of novel glycomimetics. Therefore, this work presents the synthesis of monodisperse glycooligomers carrying different sugar ligands at well-defined positions along the backbone using for the first time sequential click chemistry and stepwise assembly of functional building blocks on solid support. This approach allows for straightforward access to sequence-defined, multivalent glycooligomers with full control over number, spacing, position, and type of sugar ligand. We demonstrate the synthesis of a set of heteromultivalent oligomers presenting mannose, galactose, and glucose residues. All heteromultivalent structures show surprisingly high affinities toward Concanavalin A lectin receptor in comparison to their homomultivalent analogues presenting the same number of binding ligands. Detailed studies of the ligand/receptor interaction using STD-NMR and 2fFCS indeed indicate a change in binding mechanism for trivalent glycooligomers presenting mannose or combinations of mannose and galactose residues. We find that galactose residues do not participate in the binding to the receptor, but they promote steric shielding of the heteromultivalent glycoligands and thus result in an overall increase in affinity. Furthermore, the introduction of nonbinding ligands seems to suppress receptor clustering of multivalent ligands. Overall these results support the importance of heteromultivalency specifically for the design of novel glycoligands and help to promote a fundamental understanding of multivalent binding modes.

Mechanical carbohydrate sensors based on soft hydrogel particles.

Elastic sensors: A simple method is presented for the measurement of specific biomolecular interactions with soft colloidal hydrogel particles (SCPs) as sensors. Carbohydrate/lectin interactions (see picture; green: carbohydrate molecules) were studied by optical detection of the mechanical deformation of the particles on a lectin surface. The affinity of various carbohydrate inhibitors could also be readily determined.