4-4-20 Anti-Fluorescyl IgG Fab' Recognition of Membrane Bound Hapten: Direct Evidence for the Role of Protein and Interfacial Structure

Abstract

The surface forces apparatus was used to identify the molecular forces that control the interactions of monoclonal 4-4-20 antifluorescyl IgG Fab' fragments with fluorescein-presenting supported planar bilayers. At long range, the electrostatic force between oriented Fab' and fluorescein monolayers was controlled by the composition of the protein exterior surrounding the antigen-combining site rather than by the overall protein charge. The measured positive electrostatic potential of the Fab' monolayer at pH > pI(Fab') was consistent with the structure of the exposed Fab' surface in which a ring of positive charge at the mouth of the antigen-combining site dominates the local electrostatic surface properties. Substantial differences in the electrostatic forces measured with denatured Fab' further demonstrated that the measured electrostatic surface properties and the consequent long-range interaction forces are controlled by the protein surface composition. At short range, the strength of the Fab'-mediated adhesion was modulated not only by the length of the fluorescein tether but also by membrane hydration. Steric hydration barriers at the membrane surface reduced the adhesion strength in proportion to their range of influence. These results provide direct evidence that long-range protein interactions with immobilized ligands are controlled by both the protein and the membrane surface compositions, while short-range, specific binding is modulated by both the protein structure and the membrane interfacial properties.

Direct molecular force measurements of multiple adhesive interactions between cadherin ectodomains

“…In all previous force measurements of protein-ligand interactions, bond failure results in the abrupt snap of the molecules out of adhesive contact (14,15,(25)(26)(27)(28)(29)(30)(31)(32). By contrast, the cadherin unbinding profile (Fig.…”

“…The aqueous subphase was maintained at 25°C and contained 0.25 M Cu(NO 3 ) 2 , 50 mM Tris buffer, and 100 mM NaNO 3 at pH 7.5. The lipid monolayer was then transferred at constant pressure onto a monolayer of gel-phase dipalmitoyl phosphatidylethanolamine (0.43 nm 2 ͞lipid) supported on freshly cleaved mica substrates as described previously (14,15). Before mounting the supported bilayers in the apparatus, the disks supporting the coated mica sheets were transferred to small beakers containing 3 ml of 3 nM cadherin, 50 mM Tris buffer, 100 mM NaNO 3 , and 1 mM Ca(NO 3 ) 2 at pH 7.5, 25°C.…”

“…1). The latter is done by draining the liquid from the apparatus and then burning off the organic layers by UV irradiation (15,18). In this case, D ϭ T 2 Ϫ 2(T DSIDA ϩ T DPPE ).…”

“…Normalizing the energy density by the surface coverage of cadherin (70 nm 2 ͞protein) on the bilayers gives an average cadherin bond energy (assuming each protein forms a transmembrane bond) of 2 kT. This is substantially weaker than, for example, antibody-antigen interactions (15). Nevertheless, these proteins aggregate 1 m beads, and cells expressing the full-length cadherin on their surfaces bind to monolayers of the CEC1-5, although less effectively than to the wild-type protein.…”

Feasibility of measuring antigen–antibody interaction forces using a scanning force microscope

“…The surface forces apparatus (SFA), a benchmark technique for accurately measuring force-distance laws on a very small scale [22], has also been used to measure specific ligand-receptor binding forces. Helm et al [23] and Leckband et al [24,25] have immobilized both streptavidin/biotin and antifluorescyl IgG/fluorescein to mica SFA surfaces using Langmuir-Blodgett lipid monolayer deposition. It was found, by normalizing over the relatively large contact area, that the rupture force between a streptavidin-biotin bond was 80 pN [24].…”