BNC
BIOPHYSICS & NANOSCIENCE CENTRE
CNISM & Tuscia University
Viterbo, Italy
Salvatore Cannistraro
www.unitus.it/biophysics/
AFM COST MEETING – Paris – May 2011
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BNC
BIOPHYSICS & NANOSCIENCE CENTRE
CNISM & Tuscia University
Viterbo, Italy
Research activities
Scanning Probe
Nanoscopies &
Spectroscopies
Surface-Enhanced
Raman Spectroscopy
Surface Plasmons
Resonance
Modelling &
Molecular
Dynamics
Biosensing &
Bioelectronics
AFM COST MEETING – Paris – May 2011
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Detector
Transducer
Molecular
recognition
element
Marker
• Reduced dimensions
• Small recognition volume
• High speed of response
• Low cost
• High resolution
• Low noise
• Signal enhancement
Biologic recognition event
Electronic
signal
Immobilization strategy
• Functionality preservation
• High charge transport efficiency
• Proper orientation
• Flexibility
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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
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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.
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Biorecognition
Biorecognition refers to highly specific interactions between two biological
molecules, exhibiting unambiguous one-to-one complementarity.
Biorecognition is involved in many important biological processes,
including genome replication and transcription, enzymatic activity,
immune response, cellular signalling, ...
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Biorecognition with
Atomic 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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Protein – proten interaction
1.
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.
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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)
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p53: the guardian of the genome
Mdm2 is the main
down-regulator of p53,
binding its N-terminal
region
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p53-Mdm2 complex: AFS results
For the first time
Full-length proteins
Single molecule study
Mdm2 is immobilized to the tip targeting Lys
residues via PEG.
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)
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Azurin and its role in cancer regression
• Azurin, a small (14kDa – 128 residues) coppercontaining 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
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p53-Azurin: a heterogeneous complex
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
(De Grandis, et al.JMR 2007)
(Taranta et al., JMR 2008)
TAD= trans activation
domain
Pro= Prolin-reach domain
TAD
p53
N1
DBD
Pro
50
64
92
102
RD
TD
292 326 353
363
393
DBD=Dna-binding domain
TD=tetramerization domain
RD=regulatory domain
NTD=N-terminal domain
NTD
Mdm2
CTD
CTD=C-terminal domain
Question: can azurin
stabilize p53, by competing
with Mdm2 for the same
binding site?
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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
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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
1
DBD
94
CD studies
CTD
292
Az
393
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 with an
anticancer azurin-derived peptide
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
The study of the p28-p53 interaction could
provide rewarding information on p28 mode of
X-ray structure
of azurin
from
action
at the molecular
level
and possibly might
Pseudomonas
help to refine aeruginosa
the initial molecule in order to
raise its anticancer potentialities
Asp77
Leu50
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
Yamada T, et al., 2009. Mol. Cancer Ther. 8: 2947–2958.
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Atomic Force Spectroscopy study
NTD
TAD= trans activation domain
DBD
CTD
Pro= Prolin-reach domain
DBD=Dna-binding domain
TAD
N1
Pro
50
64
DBD
RD
TD
92 102
292 326 353
363
393
TD=tetramerization domain
RD=regulatory domain
NTD=N-terminal domain
CTD=C-terminal domain
p28
p28
p53
p28
DBD
NTD
OH
N
OH
EtOH
NHS
EtO
EtOH
OH
Si
O
O
OHOH
OH
OH OH
H
N
EtO
Si
EtO EtO
O
NHS
O
H
N
O
APTES
APTES
N
O
O
n
O
N
nH
N
H
PEGPEG
(n=24)(n=24)
N
N
S
Cys
S
MAL
p28
Cys
p28
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.
MAL
APTES: 3-aminopropyl-triethoxysilane; NHS: N-hydroxysuccinimide; PEG: polyethylenglycol; MAL: maleimide
H2O
EtOH
EtO
OH OH
OH EtOH
O
OH
OH OH
OH O
OH
H2NO
Si
H2O
H2H
O
C
H
C
N
p53
or
p53 DBD
or
Full-length
p53 NTD
EtO
EtO
Si
EtO
APTES
EtOH
N
H
C
H
C
N
p53
GLUTARALDHEYDE
H O
H O
2
2
or p53 DBD or p53 NTD
p53 and its DBD (aa 94288), were kindly provided by National
Cancer Institute ´Regina Elena´ of
Rome; p53 NTD (aa 1-93) were supplied
by Dr. Bucciantini (University of
17
Florence).
p28
p53
p28
p28
DBD
NTD
Unbinding force= 82 pN
Unbinding force = 95 pN
koff = 0.13 ± 0.03 s-1
τ = 1/koff = 7.7 s
xβ = 0.47 ± 0.02 nm
koff = 0.012 ± 0.006 s-1
 = 1/koff = 83 s
xβ = 0.46 ± 0.05 nm
1. 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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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
H2
L3
H2
L3
L1
L2
L2
S10
DBD
S9
S10
p28Folded
DBD
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p28Unfolded
S9
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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 ligandreceptor 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
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Atomic Force Spectroscopy and
Biomolecular Recognition
Editor(s): Salvatore Cannistraro; Anna Rita Bizzarri
http://www.crcpress.com/product/isbn/9781439862377
Table of contents
Introduction
Biorecognition Processes, Dr. Bongrand, France
Atomic Force Microscopy and Spectroscopy, Dr. Hoelscher, Germany
Theoretical Models, Dr. Friddle, Usa
Immobilization Strategies, Dr. Akhremitchev, Usa
Data Analysis, Drs. Pellequer and Parot, France
Outline of the Most Relevant Applications, Drs. Bizzarri and
Cannistraro, Italy
Conclusions and Perspectives
Price: $129.95
Cat. #: K12890
ISBN: 9781439862377
ISBN 10: 1439862370
Publication Date: December 26, 2011
Number of Pages: 240
forthcoming book
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Biophysics & Nanoscience Centre
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/
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