Biological Applications of Dynamic Force Spectroscopy - Dynamic Force Spectroscopy and Biomolecular Recognition - page 229

applied force on the system at a given instant but also on the entire “history” of the
applied force. Accordingly, they suggested to revise the belief that the applied force
could simply tilt the energy barrier, and they proposed to introduce a path-dependent
analysis of the DFS data. From a general point of view, this means that the com-
monly used description of unbinding process in terms of the Bell-Evans model is
an oversimplification which, in some cases, may give rise to some inconsistencies,
as also observed for avidin-biotin pairs (see above). On such a basis, they solicited
the scientific community to develop new theoretical tools, including force-history
dependence, to analyze and interpret the DFS unbinding data of some biomolecu-
lar systems.
Li et al. (2003) have investigated the interaction between the transmembrane pro-
tein α 5 β 1 integrin with fibronectin, a glycoprotein largely present in the extracellular
matrix such a complex plays a crucial role in cell differentiation and even in the adhe-
sion of cells to the extracellular matrix. They have used a setup in which cell lines
expressing only α 5 β 1 integrin have been immobilized on the AFM tip, whereas wild
type or mutated fibronectin fragments have been immobilized on the substrate. They
have focused the attention on the role played on the interaction properties by both the
specific three aminoacid long sequence, named RGD, and the so-called synergy site
of fibronectin. The presence of two linear trends in the unbinding force as a function
of the logarithm loading rate for all the analyzed systems pointed out the existence
of two energy barriers in the energy landscape (see Table 6.5). In particular, they
suggested an inner and an outer energy barrier whose relative heights determine the
overall kinetic properties of the system. The inner barrier has been found to depend
only on the RGD sequence on fibronectin, whereas the outer one depends on both
the RGD-specific sequence and the synergy site. On such a basis, it has been sug-
gested that the presence of two barriers in the energy landscape could be a common
characteristic of molecules involved in adhesion processes, since a modulation of
their relative heights could give some flexibility in the response to external forces.
Successively, the same authors have extended their approach to study the interaction
of α 5 β 1 integrin with VCAM-1, an endothelial ligand essential for the extravasa-
tion of leukocytes, where they mediate the firm adhesion to surfaces (Zhang et al.,
2004). The recombinant α 5 β 1 integrins (wild type and mutated forms) were immo-
bilized on the AFM tip while VCAM-1 was anchored to a culture dish. Again, they
found the existence of two barriers in the energy landscape for both wild type and
mutant forms of VCAM-1 molecules. The inner barrier has been demonstrated to
give a stronger resistance to the external forces through ionic interactions between
Mg 2 + and the N-terminal domain of a α 5 β 1 subunit. This provides a molecular basis
to understand how the α 5 β 1 integrin-VCAM-1 interaction is able to resist a pulling
force that is observed to occur in nature. This work witnesses the capability of DFS
to provide remarkable insights into the mechanisms regulating the leukocyte ability
to remain adherent to the surfaces in the presence of the shear force exterted by the
bloodstream.
Dynamic force spectroscopy has been also applied to investigate complexes
involved into platelet adhesion. A key role in these processes is played by fibrinogen,