Biomedical Engineering Reference
In-Depth Information
peptide formed by 20 aminoacid residues has been derived from the DNA transcrip-
tional activator PhoB involved in phosphate metabolism; such a peptide including
the specific sequence responsible for the interaction with DNA. Besides the native
form, two different single point mutated forms (mut1 and mut2) for the peptide have
been analyzed. Preliminarily, the authors have conceived competition experiments
using the native, or one of the mutated forms of the peptide, to investigate the speci-
ficity of the interactions with DNA. They found that the wild type and two of the
three mutated forms resulted into many binding events that have been traced back to
a specific binding to the DNA sequence. Instead, the other one yielded a few binding
events, which statistically led to conclude that there is no interaction with DNA. In
addition, the wild type and two mutated forms of the peptide exhibit a linear trend
dependence for the unbinding force as a function of the logarithm of the loading rate
when they interact with DNA (see Table 6.4). The extracted dissociation rate and
the energy barrier width values are found drastically different in the various systems,
indicating that even single point mutations of the peptide can significantly modu-
late its interaction properties with DNA. Furthermore, the dissociation rate and the
energy barrier width values generally fall in the same range of those obtained for
protein-protein complexes, confirming the similarity of the dissociation properties
of these biomolecular systems. In a general way, the results put into evidence the
high sensitivity of DFS to probe the binding specificity between these molecules,
supporting even the possibility to combine DFS with chemical synthesis strategies
for selection of drugs or ligands.
Krasnoslobodtsev et al. have probed the interaction between the tetrameric pro-
tein SfiI with a double-stranded oligonucleotide from DNA (Krasnoslobodtsev et al.,
2007). Sfil belongs to a family of restriction enzymes that recognize and specifically
bind to DNA for cleavage reaction. In particular, Sfil binds two DNA duplexes, each
one containing 13 bp, separated by a 5 bp palindromic region that is apparently not
essential for the recognition. To investigate the role of this nonspecific sequence, the
authors have studied the unbinding force of Sfil interacting with one of the three
different DNA fragments, each being formed by 40 bp including the two specific
sequences and a different nonspecific sequence. For all the three systems, the unbind-
ing force measured as a function of the logarithm of the loading rate has shown a
linear trend with almost the same slope but with a different intercept. According to
the Bell-Evans model, the existence of a single barrier characterized by both dif-
ferent dissociation rates and energy barrier widths has been derived (see Table 6.4).
The rather high dissociation rate values observed for all the cases suggest that the
complexes possess a transient character. Interestingly, the DFS measurements have
been coupled with a topographic investigation by AFM imaging and it was found
that the complexes are quite stable, irrespectively of the corresponding high dissoci-
ation rate value. To reconcile such an apparent inconsistency, the authors have sug-
gested that the complex transiently dissociates while, however, the two biomolecules
remain in close proximity. The significantly different dissociation rate values found
for the three various complexes point out that the stability of each complex can be
modulated by acting on a small portion of the DNA sequence that was assumed not
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