Post on 11-Jan-2016
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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
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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
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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
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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
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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)
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Biorecognition refers to highly specific interactions between two biological molecules, exhibiting unambiguous one-to-one
complementarity.
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BiorecognitionBiorecognition
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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
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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.
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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
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p53: the guardian of the p53: the guardian of the genomegenome
Mdm2 is the maindown-regulator of p53, binding its N-terminal
region
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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)
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p53-Mdm2 complex: AFS resultsp53-Mdm2 complex: AFS results
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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.
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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)
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p53-Azurin: a heterogeneous p53-Azurin: a heterogeneous complexcomplex
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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
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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
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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.
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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
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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
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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.
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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
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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
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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
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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