Conclusions and Perspectives - Dynamic Force Spectroscopy and Biomolecular Recognition - page 244

interactions between biomolecules. The use of suitable theoretical models is, on the
other hand, essential to analyze DFS data since the unbinding properties of the part-
ners are measured in nonequilibrium conditions under the application of an external
force. It should be remarked that the application of the external force, yielding a
lowering of the lifetime of the biological pairs, makes accessible the investigation of
complexes characterized by extremely long lifetimes, such as avidin-biotin whose
dissociation process does not take place spontaneously in a reasonable observation
time. In general, the Bell-Evans model derived in the theoretical framework of a ther-
mal escape over an energy barrier under the effect of an external force (see Chapter 3)
has provided a good description of the unbinding force trend as a function of the log-
arithm of the loading rate, for the largest part of the analyzed biological systems,
allowing to extract the dissociation rate and the barrier width of the energy land-
scape. However, the recurrent discrepancies found in the results from well-defined
biological systems has recently stimulated the development of a more general theo-
retical description of the unbinding processes, even to reach a deeper understanding
of biorecognition. In this respect, some important factors such as (1) the possible
deformation of the energy landscape upon the application of the external force, (2)
the relationship between the cantilever spring constant and the intermolecular poten-
tial stiffness, (3) the occurrence of rebinding processes between the partners, (4) the
partial unfolding of the biomolecules upon the pulling force, (5) the applied force
direction with respect to the reaction coordinate, should be taken into account by
more comprehensive models. Along this direction, the Jarzynski theoretical model,
which allows to evaluate the binding free energy, from the irreversible work done
along nonequilibrium paths from the bound to the unbound state, constitutes an inter-
esting novelty susceptible to enlarge the amount of information that can be obtained
by DFS experiments on biological systems.
A crucial role is also played by the setup used to perform the DFS unbind-
ing experiments. In particular, the methodologies followed to immobilize the
biomolecules to the AFM surfaces (substrate and tip) should fulfill some impor-
tant requirements: (1) Only two biomolecular partners should be possibly involved
in the biorecognition process; (2) A reliable discrimination between specificand
nonspecific unbinding events should be facilitated; and (3) The preservation of both
the native structure and the functionality of the biomolecules has to be ensured (see
Chapter 4). In general, an appropriate use of flexible linkers connecting the biomolec-
ular partners to the inorganic surfaces, and undergoing a controlled stretching dur-
ing the unbinding process, in most cases satisfy these conditions, especially when
it is combined with suitable theoretical models or computer simulations. In spite of
the large efforts made by the DFS community to continuously develop and imple-
ment new immobilization protocols, a definite strategy that could permit to standard-
ize the experimental features and the corresponding data analysis is still lacking. It
should, however, be kept in mind that the most appropriate procedure to immobi-
lize biomolecules should be chosen according to the specific features of the system
under analysis and to the information that should be extracted from the DFS data.
For example, whole cells, or part of them, should be directly used in the DFS setup