1

www.unitus.it/biophysics/www.unitus.it/biophysics/

BIOPHYSICS & NANOSCIENCE CENTREBIOPHYSICS & NANOSCIENCE CENTRECNISM & Tuscia UniversityCNISM & Tuscia University

Viterbo, ItalyViterbo, Italy

BNCBNC

AFM COST MEETING – Paris – May 2011

Salvatore CannistraroSalvatore Cannistraro

2

Research activitiesResearch activities

AFM COST MEETING – Paris – May 2011

Scanning ProbeNanoscopies & Spectroscopies

Surface-EnhancedRaman Spectroscopy

Modelling & Molecular Dynamics

Biosensing & Bioelectronics

Surface Plasmons Resonance

BIOPHYSICS & NANOSCIENCE CENTREBIOPHYSICS & NANOSCIENCE CENTRECNISM & Tuscia UniversityCNISM & Tuscia University

Viterbo, ItalyViterbo, Italy

BNCBNC

3

MarkerMolecularrecognitionelement

Biologic recognition event

Transducer

Det

ecto

r

Electronicsignal

• 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

Current resolution limit (ELISA): 10-10 M, 1013 molecules

Ideal goal for early diagnosis:detection of a single moleculedetection of a single molecule

Main limit: NOISENOISE

Concrete goal: resolution limit to 10-18 M, 105 molecules

Such result can be achieved by improving biosensors with:

Molecular biorecognition exploitation: Molecular biorecognition exploitation: one signal per eventNanostructures conjugation: Nanostructures conjugation: noise lowering

and signal enhancement

AFM COST MEETING – Paris – May 2011

5

Nanotechnological structures: nanoparticles,

nanotubes etc.

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)

AFM COST MEETING – Paris – May 2011

66

Biorecognition refers to highly specific interactions between two biological molecules, exhibiting unambiguous one-to-one

complementarity.

AFM COST MEETING – Paris – May 2011

BiorecognitionBiorecognition

7AFM COST MEETING – Paris – May 2011

Biorecognition with Biorecognition with

Atomic Force SpectroscopyAtomic Force Spectroscopy

Bizarri AR, Cannistraro S (2009) J. Phys. Chem. B 113: 16449-16464

Bizzarri AR, Cannistraro S. (2010) Chem. Soc. Rev. 39: 734-749

88

2. 2. p53-Mdm2:p53-Mdm2: a stable complex, with low koff, formed by a human tumor suppressor and its down-regulator.

3. 3. p53-Azurin: p53-Azurin: a a bacterial protein showing an anti-cancer action forms a heterogeneous complex with p53.

4. 4. p28-p53:p28-p53: an azurin-derived peptide fragment an azurin-derived peptide fragment displaying the same displaying the same anticancer potentiality of the anticancer potentiality of the whole protein.whole protein.

AFM COST MEETING – Paris – May 2011

Protein – proten Protein – proten interactioninteraction

1.1. Azurin-Cytochrome c551: Azurin-Cytochrome c551: a transient complex, with high koff , where the linking spacer may play a crucial role.

99

Biological interest: Biological interest: electron transfer electron transfer interaction interaction involved in the nitrate respiration of bacterium Pseudomonas aeruginosa. First AFS study on an electron transfer complex

ImmobilizationImmobilization strategies: 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:AFS results:• Single energy barrier;• koff values consistent with a transient complex: 7 and 14 s-1;• immobilization via organic spacers increases the binding efficiencyBonanni et al., BJ 89, 2783 (2005) and JPCB 110, 14574 (2006)

Docking simulations:Docking simulations: best complex from close contact between the hydrophobic regions of the two proteinsOptim. ET [Bizzarri et al., JMR 20, 122 (2007)]

Azurin-Cythocrome c551: a Transient Azurin-Cythocrome c551: a Transient ComplexComplex

AFM COST MEETING – Paris – May 2011

10

p53: the guardian of the p53: the guardian of the genomegenome

Mdm2 is the maindown-regulator of p53, binding its N-terminal

region

10AFM COST MEETING – Paris – May 2011

1111

Full-length proteinsSingle molecule study

Mdm2 is immobilized to the tip targeting Lys residues via PEG.

p53 is anchored to a gold substrate through - cisteamine - glutaraldehyde.

For the first time

koff = (1.5 ± 0.5) s-1 t off = (0.7 ± 0.2) s

Possible transient interaction (regulative action)

AFM COST MEETING – Paris – May 2011

p53-Mdm2 complex: AFS resultsp53-Mdm2 complex: AFS results

12

Azurin and its role in cancer Azurin and its role in cancer regressionregression

• 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.

AFM COST MEETING – Paris – May 2011

• The specificity of the Azurin-p53 interaction has been studied at single molecule level

Unbinding strength: 75 pN

koff = 9 ·10-2 s-1

Taranta M, Bizzarri AR, Cannistraro S (2008) J. Mol. Recognit. 21: 63–70.

1313

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).

Pro DBD TD RD

N1 1029264 292 326 353 36350

NTD CTD

TAD

393

p53p53

AzurinAzurin

Mdm2Mdm2

Question: can azurin stabilize p53, by competing

with Mdm2 for the same binding site?

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

(De Grandis, et al.JMR 2007)(Taranta et al., JMR 2008)

AFM COST MEETING – Paris – May 2011

p53-Azurin: a heterogeneous p53-Azurin: a heterogeneous complexcomplex

1414AFM COST MEETING – Paris – May 2011

Ternary complex p53/Mdm2/AzurinTernary 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 a ternary complexcomplex

15AFM COST MEETING – Paris – May 2011

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.

NTD DBD CTD

AzAz

1 94 292 393

CD studiesCD 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

16

Molecular interaction of p53 and its domains Molecular interaction of p53 and its domains with an anticancer azurin-derived peptide with an anticancer azurin-derived peptide

Leu50

Asp77

p28p28LSTAADMQGVVTDGMASGLDKDYLKPDD

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

X-ray structure of azurin from Pseudomonas aeruginosa

This antitumour activity is connected with the ability of p28 to bind to p53.

p28 shows antiproliferative activities against a number of cancerous cells.

Yamada T, et al., 2009. Mol. Cancer Ther. 8: 2947–2958.

AFM COST MEETING – Paris – May 2011

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

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

17

p28

p53

p28

DBD

Atomic Force Spectroscopy studyAtomic Force Spectroscopy study

p28

NTD

PEG (n=24) MALAPTES

APTES GLUTARALDHEYDE

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.

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).

APTES: 3-aminopropyl-triethoxysilane; NHS: N-hydroxysuccinimide; PEG: polyethylenglycol; MAL: maleimide

DBD

Pro DBD TD RD

N1 1029264 292 326 353 36350

NTD CTD

TAD

393

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

18

p28

p53

p28

DBD

p28

NTD

Unbinding force = 95 pN

koff = 0.012 ± 0.006 s-

1

= 1/koff = 83 s

xβ = 0.46 ± 0.05 nm

Unbinding force= 82 pN

koff = 0.13 ± 0.03 s-1

τ = 1/koff = 7.7 s

xβ = 0.47 ± 0.02 nm1. A specific biorecognition process occurs between p28 and full length p53 leading to the formation of a stable p53-p28 complex.

2. Within p53, p28 binds to its DBD while almost no interaction has been found between p28 and the NTD.

3. The DBD-p28 complex is more stable than the p53-p28, having a ten times longer lifetime.

19

H2

S10

L2

L3

S9

p28Folded

DBD

H2

p28Unfolded

L1

L2

L3

DBDS9

S10

Folded p28-DBD interaction Unfolded p28-DBD interaction

AFM COST MEETING – Paris – May 2011

The p28-DBD interaction has been also confirmed and modelled by means of computational docking and molecular modelling procedure

2020

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 azurinAFM COST MEETING – Paris – May 2011

21

Atomic Force Spectroscopy and Biomolecular Recognition

Editor(s):  Salvatore Cannistraro;  Anna Rita Bizzarri

Table of contents

IntroductionBiorecognition 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

http://www.crcpress.com/product/isbn/9781439862377

forthcoming book

2222

Anna Rita Bizzarri(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/www.unitus.it/biophysics/

Biophysics & Nanoscience Centre

AFM COST MEETING – Paris – May 2011