PROBING CELLULAR ADHESION AT THE SINGLE-MOLECULE LEVEL - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 235

11.3.2 Force Measurements

Any of the conigurations described in the previous section can be used to
carry our force measurements on single receptor-ligand complexes and
various approaches have been considered. The irst approach consisted in
measuring the forces required to break the bond when pulled apart. 28,30,47
However, because of thermal agitation and the statistical nature of bond
dissociation, this force is not unique, but spread, and depends on the rate
of force application. 48,49 The current approaches have the inal goal of
characterizing the free energy landscape of the interaction in terms of the
intrinsic dissociation lifetime at zero force ( τ 0 ), and the potential width ( γ )
and height (

$ G ‡ ). More importantly, the characterization of the interaction
will provide a description on how force affects the lifetime of the bond. The
theoretical framework is based on the idea that a mechanical force will
distort the energy landscape of the interaction, lowering the energy barrier
and facilitating dissociation. This concept was originally applied to biological
bonds by Bell in 1978. 50 In this section, we will describe the experimental
approaches used to characterize biological interactions.

11.3.2.1 Dynamic force spectroscopy

DFS is possibly the most widely used approach to characterize the adhesion
strength of biological bonds. The adhesion strength of several biological
complexes has been measured using DFS, including eukaryotic receptors,
such as cadherins, integrins and selectins, bacterial receptors, such as FimH
and mucin, and even virus proteins. 5-8,26,37,39,51-54 In addition, it has been also
used to determine the unfolding kinetics of various proteins.

55,56

The approach

consists in measuring the rupture forces ( f r ) of receptor-ligand interactions

by applying a constant loading rate, i.e. rate at which force is applied ( r f ) ( Fig.

11.4 ; for practical issues, it is sometimes useful to use force-time, instead
of force-distance, curves since the slope before rupture is a direct estimate
of the applied loading rate). At a given loading rate and because of thermal
agitation, rupture forces are not unique but follow a certain probability
distribution 48,57 ( Fig. 11.5b ) . The situation is even more complicated as the
rupture force will also depend on the dynamics of loading, given the statistical
nature of the dissociation kinetics. As mentioned before, biological bonds can
be characterized by an intrinsic lifetime at zero force ( τ 0 ), which is the inverse
of the characteristic rate at which the complex spontaneously dissociates, i.e.
the dissociation rate ( k off , the bond resists

detachment, giving rise to a measurable force. 10,49,53 As a result, the most

k off = 1/ τ 0 ). If pulled faster than