Biomedical Engineering Reference
In-Depth Information
of the two available arms of the antibody interacting with a methylated DNA site.
During the retraction process, the complexes involving the two arms of the antibody
break in sequence from their corresponding ligands; the measurement of the PEG
stretching allows to estimate the distance between the two methylated groups. Such a
nanomechanical biorecognition-based approach made it possible to determine, there-
fore, a structural parameter of DNA whose characterization was not accessible by
other methods. Moreover, the approach is suitable to be implemented in bionanode-
vices for high-throughput measurements of DNA chips to obtain posttranscriptional
information on DNA.
The advent of AFM equipment with low-instrumental drift allows to follow the
dynamical behavior of systems for a rather long time, opening, thus, the possibility to
investigate aspects of biomolecular systems otherwise hidden or obscured by other
processes. Very recently, Junker et al. have exploited the high sensitivity of DFS
combined with high temporal stability to study conformational changes of calmod-
ulin, which is one of the most prevalent Ca
2
+
signaling proteins in eukariotic cells
(Junker et al., 2009). Notably, calmodulin can bind to more than 300 different target
proteins to regulate numerous calcium-dependent functions. To study the confor-
mational changes of calmodulin by DFS, the authors have sandwiched calmodulin,
formed by two different domains, between the AFM tip and the substrate by using
immunoglobulins. The application of a loading rate much lower in comparison to
that usually used in standard DFS works has allowed them to follow the fluctua-
tions of calmodulin in real time. An analysis of these fluctuations has shown that the
calmodulin undergoes a folding-unfolding transition upon varying the Ca
2
+
concen-
tration upon binding specific target peptides. These results witness that watching a
single molecule at a time by DFS enable to follow molecular processes that would
be lost in macroscopic measurements averaged over large numbers of molecules. On
the other hand, the ability of DFS to watch folding or binding dynamics in real time
could pave the way to direct observations of molecular reaction mechanisms as a
sequence of structural events along microscopic pathways.
6.5 CONCLUSIONS
During the last years, DFS has become a progressively more rewarding and refined
tool to investigate biomolecular systems. As it emerges from the examples discussed
in the previous sections, DFS has been applied to a wide variety of biological com-
plexes at the basis of many vital functions. Its capability to measure picoNewton
unbinding forces between a single couple of biomolecules, in near-physiological
conditions and without labeling, warrants enormous potentialities to deepen the
knowledge of these systems, well-complementing information from traditional bio-
chemical and spectroscopic techniques. It is now well ascertained that the knowledge
of the unbinding force between two biomolecules does not directly provide a quan-
titative evaluation of the strength of the complex they may form. Indeed, quite dif-
ferent biomolecular systems, such as antigen-antibody, protein-ligand complexes,
DNA-based systems, exhibit rather similar values for the unbinding force measured
at the same loading rate. However, from a collection of unbinding force values,
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