Force Spectroscopy

Besides imaging surfaces, the AFM can be also applied to study interaction forces between tip and sample. In this case, the deflection of the cantilever is measured as the distance between the probe and the sample is varied, thus generating force-distance curves. Such working configuration is called force spectroscopy. With these so-called force-distance curves it is possible to measure intermolecular forces: by using a measuring tip with receptors bound and recording force vs. distance cycles on surface-bound ligands, unbinding forces of ligand-receptor pairs can be probed at the level of single molecule.

Recognition events between the partners have been observed, as revealed by characteristic pull-off jumps in the force-distance retraction curve.

By this technique it is also possible to investigate the complex dissociation kinetics, by monitoring the unbinding forces as a function of the scan rate.

We have used force spectroscopy to study the interaction between redox partners, as Cytochrome C551 and Azurin, to investigate the binding between the tumor suppressor protein p53 and the bacterial protein Azurin, and eventually to study the interaction between the p53 and its domains ans the anticancer azurin-derived peptide p28.

Azurin-Cytochrome complex has been studied by coupling Cytochrome C551 to the tip via a long flexible spacer molecule, and by directly immobilizing Azurin on a flat gold substrate, thanks to the presence of exposed cysteine residues, via the formation of S-Au bonds [Bonanni et al., Biophysical Journal 2005]. Subsequently, the molecular recognition between these two proteins has been examined after optimizing Azurin adsorption on gold via sulfhydryl terminated alkanethiol spacers [Bonanni et al., Journal of Physical Chemistry B 2006]. The introduction of this linker demonstrated to favour the complex formation, as evidenced by the decreasing of the dissociation rate constant, from 14 s^-1 (for Azurin directly adsorbed on gold) to 6.7 s^-1.

Our results, obtained from the analysis of single electron-transfer proteins, immobilized on a metal substrate upon two different techniques, are good starting points for possible detection of single recognition events as an electric signal, with potential applications in ultra-sensitive bio-nanodevices designed for biological screening.

We have focused our attention to the study of the p53/azurin complex. Indeed the understanding of the molecular mechanisms, determining p53 stabilization further to Azurin binding, may be useful for developing new targeted anticancer strategies based on this complex formation.

p53-Azurin complex has been investigated by immobilizing p53 on a gold surface via a flexible poly(ethylene glycol) (PEG) linker, and by tethering Azurin to the tip via a sulfhydryl terminated spacer. This work allowed us to observe recognition events between the proteins and to establish that the complex is substantially stable, displaying a lifetime of about 11 s.

p53-Mdm2-Azurin competitive experiments

It has been demonstrated that azurin anticancer activity is connected with its interaction with p53 that leads to both the stabilization and intracellular level rise of the transcription factor. We have used AFS to explore the appealing hypothesis that azurin could compete with the main down-regulator of p53, the oncoprotein Mdm2, for the same p53 binding site thus reducing its activity. We first measured the unbinding frequency between a p53-functionalized substrate and an azurin-functionalized tip and then blocked the substrate with an azurin solution. A dramatic reduction of the unbinding frequency has been observed, confirming the specificity of the p53/azurin interaction. Parallel, we incubated the p53 sample with a solution containing Mdm2 molecules and observed that the frequency of interaction remained substantially unchanged. We thus performed a second blocking experiment: we measured the unbinding frequency between immobilized p53 and Mdm2 and then blocked the substrate with Azurin. Again, the unbinding frequency between p53 and Mdm2 was not affected by Azurin. We thus advanced the hypothesis that azurin and Mdm2 interact with two different regions of p53 and that a ternary complex is possible. The occurrence of a p53-Mdm2-azurin complex has opened a possible new scenario for the anticancer action of azurin by also providing evidence that AFS offers a valid tool for the study ternary complexes at single-molecule level.

p53 - p28 and p53 fragments/p28 complexes have been investigated by immobilizing p53 or its fragments on glass substrates via specific spacer and anchoring p28 to the tip via a PEG linker. This work allowed us to observe specific biorecognition process between p28 and both p53 and its core domain and to found that the p53 core domain/p28 complex is ten times more stable than the p28/p53 one with its 80 s half life.

With this last work we have thus given a very significant contribution in the research on the possible mechanism of action of the very new anticancer peptide p28.

If AFS allows the direct determination of the unbinding force of the biorecogniton process and of its dissociation rate constant (k_off), to completely elucidate the kinetic of a biorecognition events, a Surface Plasmons Resonance apparatus can be used.