Force-Induced Dissociation of Single Protein A−IgG Bonds

Abstract

We investigated the force-induced dissociation of single specific bonds between biomolecules. For these experiments we used a micropipet-based picoforce transducer. It consisted of a red blood cell tensed by micropipet suction. The stiffness of this "spring" was tuned by suction pressure. The proteins under investigation were immobilized onto microbeads. One type of bead carried the adhesion proteins and was biochemically attached to the red blood cell membrane. The second type, carrying the respective ligand, was held in a second micropipet which was moved by a piezoelectric transducer. Complementary beads were repeatedly brought into contact in order to form and break bonds. Yield forces exceeding 3 pN could be detected. For protein binding, the microbeads were covalently coated with a hydrogel of several nanometer thickness onto which the proteins were bound. This preparation resulted in a low frequency of nonspecific interactions between the beads. Linkages between beads and proteins were of sufficient strength for mechanical experiments on single molecules. We controlled the number of available binding sites by competitive blocking with soluble ligand so that only single molecular bonds were present between the microbeads. Protein A-IgG bonds were studied. Here we found a marked dependence of bond strength on the rate of force application. This effect is due to a force dependent rate of bond dissociation.

Unbinding process of adsorbed proteins under external stress studied by atomic force microscopy spectroscopy

“…The values of 0 and f 0 relative to the different experiments are given in Table 1. To get an idea of the sensitivity of the value of 0 to our experimental data, we represent in found for the dissociation rate in the case of various specific interactions of the type ligand͞receptor (9,16).…”

“…As a consequence, Evans (15) showed that the rupture force measured in AFM spectroscopy should depend on the retraction rate. This was clearly demonstrated for ligand͞ receptor systems over the last year (7,9). A change of the retraction rate in AFM experiments thus gives access to the dynamics of proteins.…”

“…1 should be changed into 0 g( f ), where g( f ) is related to the force-depending width of the energy barrier. However, this often constitutes a second order effect when compared with the variations of ( f ) because of the changes in exp( f͞f 0 ), and most of the unbinding experiments are interpreted within the framework of the Bell model defined by Expression 1 (9,14,16). In force measurement experiments as they are carried out with AFM, the applied force f is not constant, but increases linearly with time t: f ϭ r f t, where r f represents the constant loading rate.…”

“…We could also follow the anchoring process of the protein on the surface leading to an increase of the measured rupture forces with the protein͞surface interaction time. These new investigation tools working at the molecular level have, however, up to now, been applied mainly to the investigation of ligand͞receptor interactions (3)(4)(5)(6)(7)(8)(9)(10)(11), and only very few studies concern the area of nonspecific interactions (12,13). Moreover, these techniques are still under development, and the precise physical meaning of the measured quantities is still not fully clear.…”

Mechanically enforced bond dissociation reports synergistic influence of Mn2+ and Mg2+ on the interaction between integrin α7β1 and invasin

“…We adjusted and optimized the immobilization of proteins on microbeads previously described in (Lofas and Johnsson, 1990;Johnsson et al, 1991) including some later adaptations (Strigl et al, 1999). The three main steps are bead dextranization, dextran carboxylation, and protein immobilization.…”