Effect of Compressive Force on Unbinding Specific Protein–Ligand Complexes with Force Spectroscopy

Abstract

Atomic force microscopy (AFM) is used extensively for the investigation of noncovalent molecular association. Although the technique is used to derive various types of information, in almost all instances the frequency of complex formation, the magnitude of rupture forces, and the shape of the force-distance curve are used to determine the behavior of the system. We have used AFM to consider the effect of contact force on the unbinding profiles of lactose-galectin-3, as well as the control pairs lactose-KDPG aldolase, and mannose-galectin-3, where the interacting species show negligible solution-phase affinity. Increased contact forces (>250 pN) resulted in increased probabilitites of binding and decreased blocking efficiencies for the cognate ligand-receptor pair lactose-G3. Increased contact force applied to two control systems with no known affinity, mannose-G3 and lactose-KDPG aldolase, resulted in nonspecific ruptures that were indistinguishable from those of specific lactose-G3 interactions. These results demonstrate that careful experimental design is vital to the production of interpretable data, and suggest that contact force minimization is an effective technique for probing the unbinding forces and rupture lengths of only specific ligand-receptor interactions.

Specific Binding at the Cellulose Binding Module–Cellulose Interface Observed by Force Spectroscopy

“…The frequency histograms were fit to an inverse Gaussian distribution function to determine statistically significant forces and lengths. The mean Ni-dependent rupture force was 121 ± 7 pN at a retraction velocity of 200 nm s –1 (The retraction velocity corresponds to a loading rate of ∼0.4 nN s –1 , as calculated for the presented molecular system by Bowers et al), and the mean rupture length was 15.5 ± 0.4 nm; these values agree well with a molecular system length of about 13 nm (3 nm peptide + 10 nm PEG 24 ).…”

“…As a result, the analysis of force spectroscopy data is subject to errors associated with surface vibrations and tip fluctuations that can alter rupture morphology and lead to inconsistent identification of ruptures. Other experimental parameters, such as contact force and dwell time, can also alter rupture morphologies and impact the probability of observing rupture events, even within a single data set . To minimize these effects, we rely on automated protocols developed in our laboratory previously for the objective analysis of rupture data.…”

“…To probe this hypothesis, we exchanged MCC with the similarly sized lectin murine galectin-3 (G3, 35 kDa), a protein with no known affinity for cellulosic substrates. We have previously detailed the specific affinity ( K a ∼ 6400 M –1 ) of G3 for small galactose-based carbohydrates and demonstrated its suitability for force spectroscopy experiments. ,,, If the cellulose NC blocking agent does irreversibly inhibit nonspecific protein–cellulose interactions, we should initially observe G3–cellulose interactions blockable by the NC suspension, but binding should not reappear after removal of the NCs from the fluid cell.…”

Nanomechanical and Thermophoretic Analyses of the Nucleotide-Dependent Interactions between the AAA+ Subunits of Magnesium Chelatase

“…34 To put the ChlI–ChlD unbinding force (measured at a loading rate of about 75 nN s –1 ) in a clearer context, we can compare it with previously published unbinding forces, measured at similar loading rates: 90–100 pN for single cohesin–dockerin unbinding event, 39 177 pN for streptavidin–biotin unbinding, 40 and 60 pN for the lactose–galectin-3 complex unbinding. 41 We can conclude that the ChlI–ChlD unbinding force is consistent with the unbinding forces measured for other biologically relevant (and of comparable size) high-affinity ligand–receptor pairs that form stable complexes.…”

Targeting label free carbohydrate–protein interactions for biosensor design

“…43 Bowers and co-workers investigated the effect of applied contact force on the probability of observing rupture events, the normalized number of blockable rupture events per pull, and the rupture force and length distribution in force versus extension plots. 44 AFM tips were made from amino functionalized silicon nitride conjugated to a PEG maleimide, which were further covalently bound to thiopentyl lactoside, and thiopentyl mannoside (Fig. 6a).…”