Probing the interaction between p53 and the bacterial protein azurin by single molecule force spectroscopy.
p53 is a human tumour suppressor which regulates multiple cellular processes, including cell growth, genomic stability and cell death. Recent works have demonstrated the bacterial redox protein azurin to enter cancer cells and induce apoptosis through p53 stabilization, resulting in a tumour growth regression. Azurin has been shown to bind p53 although many details of the complex formed by these two proteins are still poorly characterized. Here, we get insight into the kinetics of this complex formation, by exploring the interaction between p53 and azurin in their environment by single molecule force spectroscopy. To this aim, azurin has been linked to the atomic force microscope tip, whereas p53 has been immobilized onto a gold substrate. Therefore, by performing force-distance cycles we have detected specific recognition events between p53 and azurin, displaying unbinding forces of around 70 pN for an applied loading rate of 3 nN s(-1). The specificity of these events has been assessed by the significant reduction of their frequency observed after blocking the p53 sample by an azurin solution. Moreover, by measuring the rupture force as a function of the loading rate we have determined the dissociation rate constant of this complex to be approximately 0.1 s(-1). Our findings are here discussed in connection with results obtained in bulk experiments, with the aim of clarifying some molecular details of the p53-azurin complex that may help designing new anticancer strategy.
Taranta
Monia
M
Biophysics and Nanoscience Centre, CNISM, Facoltà di Scienze, Università della Tuscia, Largo dell'Università, 01100 Viterbo, Italy.
Bizzarri
Anna Rita
AR
Cannistraro
Salvatore
S
D001400:Azurin / Q000737:chemistry / Q000378:metabolism*; D001696:Biomechanical Phenomena; D006046:Gold; D006801:Humans; D007700:Kinetics; D018625:Microscopy, Atomic Force; D011092:Polyethylene Glycols / Q000378:metabolism; D011485:Protein Binding; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D016159:Tumor Suppressor Protein p53; D001400:Azurin; D011092:Polyethylene Glycols; D006046:Gold