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1. BNC BIOPHYSICS & NANOSCIENCE CENTRE CNISM & Tuscia University Viterbo, Italy Salvatore Cannistraro www.unitus.it/biophysics/ AFM COST MEETING – Paris – May 2011

2. BNC BIOPHYSICS & NANOSCIENCE CENTRE CNISM & Tuscia University Viterbo, Italy Scanning Probe Nanoscopies & Spectroscopies Surface-Enhanced Raman Spectroscopy Surface Plasmons Resonance Modelling & Molecular Dynamics Biosensing & Bioelectronics Research activities AFM COST MEETING – Paris – May 2011

3. Biorecognition-based NanoBiosensors Molecular recognition element Transducer Detector Biologic recognition event Electronic signal Marker • Reduced dimensions • Small recognition volume • High speed of response • Low cost • High resolution • Low noise • Signal enhancement • Immobilization strategy • Functionality preservation • High charge transport efficiency • Proper orientation • Flexibility AFM COST MEETING – Paris – May 2011

4. Biosensing Current resolution limit (ELISA): 10-10 M, 1013 molecules Ideal goal for early diagnosis: detection of a single molecule Main limit: NOISE Concrete goal: resolution limit to 10-18 M, 105 molecules Such result can be achieved by improving biosensors with: Molecular biorecognition exploitation: one signal per event Nanostructures conjugation: noise lowering and signal enhancement AFM COST MEETING – Paris – May 2011

5. Single molecule techniques for early diagnostics • Single molecule techniques: • Atomic Force Microscopy and Spectroscopy (AFM & AFS) • Scanning Tunnelling Microscopy (STM) • Conductive-AFM • Surface Enhanced Raman Spectroscopy (SERS) Recognition capability of biomolecules (1:1) Nanotechnological structures: nanoparticles, nanotubes etc. AFM COST MEETING – Paris – May 2011

6. Biorecognition Biorecognition refers to highly specific interactions between two biological molecules, exhibitingunambiguousone-to-onecomplementarity. Biorecognition is involved in many important biological processes, including genome replication and transcription, enzymatic activity, immune response, cellular signalling, ... 6 AFM COST MEETING – Paris – May 2011

7. Biorecognition with Atomic Force Spectroscopy Bizarri AR, Cannistraro S (2009) J. Phys. Chem. B113: 16449-16464 Bizzarri AR, Cannistraro S. (2010) Chem. Soc. Rev. 39: 734-749 AFM COST MEETING – Paris – May 2011

8. Protein – proten interaction • Azurin-Cytochrome c551: a transient complex, with high koff , where the linking spacer may play a crucial role. 2. p53-Mdm2: a stable complex, with low koff, formed by a human tumor suppressor and its down-regulator. 3. p53-Azurin: a bacterial protein showing an anti-cancer action forms a heterogeneous complex with p53. 4. p28-p53: an azurin-derived peptide fragment displaying the same anticancer potentiality of the whole protein. AFM COST MEETING – Paris – May 2011 8

9. Azurin-Cythocrome c551: a Transient Complex Biological interest: electron transfer interaction involved in the nitrate respiration of bacterium Pseudomonas aeruginosa. First AFS study on an electron transfer complex Docking simulations: best complex from close contact between the hydrophobic regions of the two proteins Optim. ET [Bizzarri et al., JMR 20, 122 (2007)] • Immobilization strategies: • PEG molecules for flexible linking of cytochrome to the tip, targeting –NH2; • oriented azurin bonding to the Au (electr sens)substrate via disulphide bridge, with or without spacers. • AFS results: • Single energy barrier; • koff values consistent with a transient complex: 7 and 14 s-1; • immobilization via organic spacers increases the binding efficiency • Bonanni et al., BJ 89, 2783 (2005) and JPCB 110, 14574 (2006) 9 AFM COST MEETING – Paris – May 2011

10. p53: the guardian of the genome Mdm2 is the main down-regulator of p53, binding its N-terminal region 10 AFM COST MEETING – Paris – May 2011

11. p53-Mdm2 complex: AFS results Full-length proteins Single molecule study For the first time Mdm2 is immobilized to the tip targeting Lys residues viaPEG. p53 is anchored to a gold substrate through - cisteamine - glutaraldehyde. koff = (1.5 ± 0.5) s-1 t off = (0.7 ± 0.2) s Possible transient interaction (regulative action) 11 AFM COST MEETING – Paris – May 2011

12. Azurin and its role in cancer regression • Azurin, a small (14kDa – 128 residues) copper-containing protein with electron-transfer activity in Pseudomonas Aeruginosa, plays a prominent anticancer role both in vitro and in vivo (Yamada et al., 2002. PNAS 99: 14098-14103). • Azurin preferentially enters cancer cells and induces apoptosis via a caspase-mediated mitochondrial pathway. • This antiproliferative action is connected with the formation of a complex with p53. • The specificity of the Azurin-p53 interaction has been studied at single molecule level Taranta M, Bizzarri AR, Cannistraro S (2008) J. Mol. Recognit. 21: 63–70. Unbinding strength: 75 pN koff = 9 ·10-2 s-1 AFM COST MEETING – Paris – May 2011

13. p53-Azurin: a heterogeneous complex DBD TAD Pro RD TD 393 N1 50 64 92 102 292 326 353 363 NTD CTD Several experimental or computational studies have hypothesized a direct contact of Az with an undetermined binding site located in the DBD of p53 (Punj et al., 2004; Bizzarri et al., 2009; De Grandis et al., 2007) or in its NTD (Apiyo et al., 2005; Taranta et al., 2009). Azurin Question: can azurin stabilize p53, by competing with Mdm2 for the same binding site? (De Grandis, et al.JMR 2007) (Taranta et al., JMR 2008) TAD= trans activation domain Pro= Prolin-reach domain DBD=Dna-binding domain TD=tetramerization domain RD=regulatory domain NTD=N-terminal domain CTD=C-terminal domain p53 Mdm2 13 AFM COST MEETING – Paris – May 2011

14. Ternary complex p53/Mdm2/Azurin NO COMPETITION Az and Mdm2 do not compete for the same binding site of p53 and they are engaged in a ternary complex AFM COST MEETING – Paris – May 2011 14

15. 1 94 292 393 Az NTD DBD CTD Surface Plasmon Resonance studies have also shown that Azurin is able to induce a weakening of the Mdm2-p53 interaction by a non competitive inhibition mechanism, which should be figured out as a long range binding regulation. CD studies Possible allosteric regulation of this azurin-induced inhibition The contribution of our centre has been crucial in disclosing the molecular and kinetic details of the Azurin-p53 interaction. It has also suggest to search for the azurin smallest peptide fragment retaining both the azurin cellular penetration ability and anti-proliferative activitya. a)Yamada T. et al., 2009. Mol. Cancer Ther. (2009) 8: 2947-2958 AFM COST MEETING – Paris – May 2011

16. Molecular interaction of p53 and its domains with an anticancer azurin-derived peptide Asp77 Leu50 p28 shows antiproliferative activities against a number of cancerous cells. This antitumour activity is connected with the ability of p28 to bind to p53. p28 has already passed the Phase I clinical trials under the Food and Drug (FAD) administration allowance, but its mode of action has not been completely elucidate yet since molecular and kinetic details of its interaction with p53 have not been clarified p28 LSTAADMQGVVTDGMASGLDKDYLKPDD It is an amphipatic peptide belonging to an α–helix (residue 54 to 67) as well as a partial β–sheet in the crystallized azurin The study of the p28-p53 interaction could provide rewarding information on p28 mode of action at the molecular level and possibly might help to refine the initial molecule in order to raise its anticancer potentialities X-ray structure of azurin from Pseudomonas aeruginosa Yamada T, et al., 2009. Mol. Cancer Ther. 8: 2947–2958. AFM COST MEETING – Paris – May 2011

17. Atomic Force Spectroscopy study NTD DBD CTD TAD Pro DBD TD RD 393 N1 50 64 92 102 292 326 353 363 p28 p28 p53 NTD PEG (n=24) APTES MAL GLUTARALDHEYDE APTES TAD= trans activation domain Pro= Prolin-reach domain DBD=Dna-binding domain TD=tetramerization domain RD=regulatory domain NTD=N-terminal domain CTD=C-terminal domain p28 DBD p29 was kindly provided by Dr. Craig W. Beattie. Department of Surgical Oncology University of Illinois College of medicin, as well as Scientific Officier of CDG Therapeutics Inc. of Chicago, USA. APTES:3-aminopropyl-triethoxysilane; NHS: N-hydroxysuccinimide; PEG: polyethylenglycol; MAL: maleimide Full-length p53 and its DBD(aa 94-288),were kindly provided by National Cancer Institute ´Regina Elena´ of Rome; p53 NTD (aa 1-93) were supplied by Dr. Bucciantini (University of Florence).

18. p28 p28 p28 DBD NTD p53 Unbinding force= 82 pN koff = 0.13 ± 0.03 s-1 τ= 1/koff= 7.7 s xβ= 0.47 ± 0.02 nm • Unbinding force = 95 pN • koff = 0.012 ± 0.006 s-1 • = 1/koff = 83 s xβ= 0.46 ± 0.05 nm • A specific biorecognition process occurs between p28 and full length p53 leading to the formation of a stable p53-p28 complex. • Within p53, p28 binds to its DBD while almost no interaction has been found between p28 and the NTD. • 3. TheDBD-p28 complex is more stable than the p53-p28,having a ten times longer lifetime.

19. H2 H2 L3 L3 L1 L2 L2 S10 p28Unfolded p28Folded S10 S9 DBD S9 DBD The p28-DBD interaction has been also confirmed and modelled by means of computational docking and molecular modelling procedure Folded p28-DBD interaction Unfolded p28-DBD interaction AFM COST MEETING – Paris – May 2011

20. Conclusions AFS represents one of the most rewarding tools for studying biological processes, allowing to measure forces acting between individual biomolecules with sensitivity of pN, in near-physiological conditions and without labelling. AFS data permit to obtain information on the dissociation rate constant koff and on the activation free energy for a single ligand-receptor pair, complementing traditional biochemical approaches. In our case, it allowed us to reach unprecedented kinetic results on the p53/mdm2 complex formed by the two full-length proteins. Moreover , it evidenced that the p53 stabilization induced by Azurin cannot be attributed to competitive binding with respect to mdm2. The occurrence of the ternary complex opens new perspective for the anti-cancer action of azurin AFM COST MEETING – Paris – May 2011 20

21. AtomicForceSpectroscopy and BiomolecularRecognitionEditor(s):  Salvatore Cannistraro;  Anna Rita Bizzarri http://www.crcpress.com/product/isbn/9781439862377 Table of contents Introduction Biorecognition Processes, Dr. Bongrand, FranceAtomic Force Microscopy and Spectroscopy, Dr. Hoelscher, GermanyTheoretical Models, Dr. Friddle, UsaImmobilization Strategies, Dr. Akhremitchev, UsaData Analysis, Drs. Pellequer and Parot, France Outline of the Most Relevant Applications, Drs. Bizzarri and Cannistraro, ItalyConclusions and Perspectives Price:  $129.95 Cat. #:  K12890 ISBN:  9781439862377 ISBN 10:  1439862370 Publication Date:  December 26, 2011 Number of Pages:  240 forthcoming book

22. Biophysics & Nanoscience Centre Anna RitaBizzarri (Professor of Physics) Ines Delfino (Research Assistant Professor) Chiara Baldacchini (CNR-CNISM Research Fellow) Samuele Raccosta (PostDoc) Fabio Domenici (PhD Student) Xian Jin Xu (Phd Student) Simona Santini (Graduated Student) www.unitus.it/biophysics/ AFM COST MEETING – Paris – May 2011 22