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BIOPHYSICS & NANOSCIENCE CENTRE

Università della Tuscia Viterbo - Italy

BNCBNC

Nanoscopia a forza atomica per lo studio dei sistemi

biologici

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AFM is a powerful tool for studying biological systems.

The AFM method is based on the detection of

FORCES with a picoNewton sensitivity.

• In near-physiological conditions • Single molecule resolution • Without any labelling • At work

AFM environment: - Air - LIquid: Liquid cells; The tip and the

sample are fully submerged in liquid;

Biology, geologic systems, corrosion, or any

surface study where a solid-liquid interface

is involved.

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• AFM is suitable to investigate the morphological properties of biological samples by scanning a sharp tip, located at the end of a cantilever spring, over a substrate. The sample is moved relative to the cantilever in three dimensions using piezoelectric ceramic. The interaction forces between the tip and sample are measured by the cantilever deflection through the reflection of a laser beam on the free end of the cantilever and reflected into a photodiode.

x

y

z

• 0.1 nm vertical resolution, pN

sensitivity

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•The measure cantilever deflections allows a computer to generate a map of surface topography.

•Its deflection is caused by the force between the tip and the sample surface,

and it is detected by a laser beam.

Hooke’s law F = -k·Δs

s

•The force most commonly associated with AFM is an interatomic force called the Van der Waals force.

The cantilever acts as a spring.

•Feedback loop keeps constant the tip-sample distance.

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Imaging modes • Contact Mode:

• Tapping Mode:

• Non-Contact mode:

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• Imaging & topography

• Atomic force spectroscopy (AFS):biorecognition

• Nanomechanical properties of cells & polymers

• Unfolding of proteins & nucleic acids.

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Topography and imaging

Erythrocytes (50x50) mm2 Monkey Liver Cell (42x42) mm2

Cells

Fibroblast Cell (5 x 5) μm2

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Monocytes (30x30) μm2

(20 x 20) μm2 (20 x 20) μm2

(20 x 20) μm2

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Spores

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Viruses

Retrotransposon Ty3

Tobacco Mosaic Virus

Moloney Mouse Leukemic Virus

A. McPherson et al. Atomic Force Microscopy Investigation of Viruses, 2011. SIF - 20.09.2012

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Proteins

Collagen fibrils (5 x 5)μm2

Aquaporin-Z J. K. H. Hörber et al. SCIENCE 2003

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0 nm

7 nm

Plastocyanin adsorbed on Au (500 x 500) nm2.

0 1 2 3 40

5

10

15h

0 = 2.3 nm

= 0.5 nm

num

ber o

f mol

ecul

es (%

)

height (nm)

(250 x 250) nm2 Height distribution as evaluated from individual cross section analysis over 772 molecules L. Andolfi et al Surface Science 2003

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Cytochrome c immobilised on Au(111)

B. Bonanni et al. CHEMPHYSCHEM 2003

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Purple Membrane Surface

Rhodopseudomonas membrane

Membranes

A. Engel et al. Nat. Am. Inc. 2000

S. Scheuring et al. J. Struct. Biol. 2007

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DNA, (0.54x0.54) μm2

Human Chromosomes (20x20) μm2

Genetic Material

DNA

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At work

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Living human vascular endothelial cells imaged in culture media. These images, collected at 30 minute intervals, reveal the movement of living cells. (65 x 65) μm2.

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Biorecognition is involved in many important biological processes, including genome replication and transcription, enzymatic activity, immune response,

cellular signalling, ...

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

BIORECOGNITION

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At equilibrium:

off

onA

k

kK

LR

RL

][

kkonon depends mainly on molecular diffusiondepends mainly on molecular diffusion koff depends on the interaction strength and energy landscape tto=o= (k(koff off ))

--11 lifetime of the process → high variability 10lifetime of the process → high variability 1066--1010--22 s s

The study of kThe study of koffoff provides significant information on the specificity of provides significant information on the specificity of the biorecognition processthe biorecognition process

KA Affinity constant koff dissociation rate kon association rate

Biorecognition is a kinetic process

A biomolecular interaction is not necessarily optimized to achieve the highest affinity but it could have been selected to reach the best results in a short time. Importance of the kinetic parameters

][]][[ RLkLRk

dt

RLdoffon

L

R

KINETIC OF BIORECOGNITION

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LIGAND

RECEPTOR

AFS allows the measure of the interaction forces, the allows the measure of the interaction forces, the evaluation of the dissociation rate evaluation of the dissociation rate koff , the estimation the estimation

of number, height and width of number, height and width of of energy barriers

for for single and and immobilized ligandligand--receptor pairs.receptor pairs.

•• Label-free. • Physiological conditions, to preserve biomolecule morphology and functionality. •• Interaction forces with pN sensitivity, (It is based on Atomic Force Microscopy).

Atomic Force Spectroscopy (AFS)

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1) The ligand-functionalized tip is approached to a surface covered by immobilized receptors 2) The cantilever begins to deflect in consequence of intermolecular repulsive forces. 3) From this point on, the cantilever exerts a pushing force on the substrate while ligand and

receptor, brought in close proximity, can interact and form a complex. 4) The direction of motion is reversed and the cantilever retracts from the surface. During this

retraction phase (continuous line) the cantilever reaches the baseline deflection and, by further increasing the tip-sample distance, it begins to bend downwards (due to the attractive interaction-force displayed by the ligand–receptor complex).

5) When the force exerted by the cantilever overcomes the stability of the complex bonds, a sudden jump in the deflection occurs, as a consequence of the complex dissociation that separates the ligand–receptor pair.

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1. Immobilization strategy

2. Force-distance curve registration: determination of Funb

3. Bell-Evans’ Model

ATOMIC FORCE SPECTROSCOPY EXPERIMENT AND DATA ANALYSIS

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AFS Immobilization Strategies

The anchoring of the biomolecules to the inorganic surface (tip/substrate) must be

stronger than the intermolecular forces holding the complex.

Covalent binding

Flexible linkers endow the biomolecules with

Mobility and

Re-orientational freedom

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0 50 100 150 200 250 3000

5

10

15

20

25

30

Fre

quency

Unbinding Force (pN)

S • k = F Hooke’s LawS • k = F Hooke’s Law

1. Measurements of the cantilever deflection S at the single rupture event 2. Calculation of the single rupture force F by applying the Hooke’s Law 3. Iteration of the force-distance cycle 4. Construction of the unbinding force distribution 5. Estimation of the most probable rupture force: Funb

tip displacement (nm)

defl

ect

ion

(nm

)

S

FORCE CURVES:

UNBINDING FORCES

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INJECTION OF FREE PARTNER

Un

bin

din

g ev

en

t fr

eq

ue

ncy

LIGAND

RECEPTOR

A significant decrease of the unbinding probability

is expected.

WASH

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In AFS experiments the rupture of the interaction bond takes place under the influence of an external force that drives the system away from the equilibrium and the activation free energy of the reaction ΔG* is lowered by a factor proportional to the applied force F:

At equilibrium, a receptor-ligand pair changes from the bound and the unbound states with a characteristic activation free energy barrier (ΔG*) and the dissociation rate constant, koff ,is given by an Arrhenius-like expression:

koff = A e(- ΔG*/kBT)

From Unbinding force to koff

ΔG*(F)= ΔG* - F xβ

The dissociation rate koff (F) depends on the applied force koff (F) = koff e

(F xβ)/(kBT) SIF - 20.09.2012

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Tkk

r xln

x

TkF

Boff

β

β

B

unb

• Under conditions of constant loading rate, the unbinding force Funb is a linear function of the natural logarithm of the loading rate and is given by the following expression:

To extract equilibrium parameters from non-equilibrium experiments the Bell-Evans’ Model is the most widely used approach in AFS experiments

• According to the model, the effect of the applied force on the energy landscape distortion increases with raising the loading rate r at which the force is applied during time

(r=dF/dt=kv).

The Bell-Evans Model

• By plotting Funb as a function of ln r, the equilibrium parameters koff and xβ can be extracted from the slope and the intercept of a linear fit.

Energy landscapes having more than one barrier will result in Funb vs. Ln r showing more than one linear parts, with different slopes (i.e., different kinetics).

xβ

ΔG*

koff

ln r (nN/s) F

un

b (p

N)

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

3. 3. p53-Azurin: the bacterial protein Azurin shows anti-cancer action; does it form a stable heterogeneous complex with p53?

4. 4. Mdm2-p53-Azurin: a ternary complex?

55. p28-p53: an azurinan azurin--derived peptide fragment displays the same derived peptide fragment displays the same anticancer potentiality of the whole protein. What about its anticancer potentiality of the whole protein. What about its interaction with p53? interaction with p53?

APPLICATION OF BIORECOGNITION

1.1. Azurin-Cytochrome c551: a transient complex, with high koff ,

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

AFS results: • Single energy barrier; • koff values consistent with a transient complex: 7 and 14 s-1 depending by the used immobilization strategy used. • immobilization via organic spacers increases the binding efficiency. [Bonanni et al., BJ 89, 2783 (2005) and JPCB 110, 14574 (2006)]

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

Azurin-Cytochrome C551: a transient complex

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p53-Mdm2-Azurin • p53 is a potent transcription factor playing a crucial role in preventing tumour

progression. Activated in response to a signal of stress or DNA damage, it induces the transcription of genes involved in apoptosis, cell cycle arrest, DNA repair.

• Mdm2 is the major down regulator of p53: it interacts with the tumour suppressor

and keeps it at low concentration.

p53 – Mdm2 interaction represents a central target for a variety of anticancer strategies with the aim at stabilizing and enhancing p53

tumour-suppression function

It has been shown that the bacterial redox protein azurin plays an anticancer role connected with its

interaction with p53

Can azurin stabilize p53,

by competing with Mdm2

for the same binding site?

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Single energy barrier and unique transition state of the reaction ; koff = (1.5 ± 0.5) s-1 t off = (0.7 ± 0.2) s

koff = (2.5 ±0.6) s-1 toff = (0.4) s Value comparable to that of the Mdm2-p53 complex (transient character)

p53-Mdm2 p53-Azurin

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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 Mdm2 Az

p53 SIF - 20.09.2012

NTD DBD CTD

AzAz

1 94 292 393

Az-p53 binding could involve either

or the DBD of p53 or the DBD of p53

an N-terminal portion, distinct to that of Mdm2 distinct to that of Mdm2

• Circular Circular dichroismdichroism studies studies suggest a possible suggest a possible allostericallosteric regulation for this regulation for this AzurinAzurin--induced inhibition.induced inhibition.

• Surface Plasmon Resonance studies have also shown that Azurin induces a weakening of the Mdm2-p53 interaction by a non competitive inhibition mechanism

Studies at our Centre have been crucial in disclosing the molecular and kinetic details of the Azurin-p53 interaction.

They have also suggested to search for the azurin smallest peptide fragment retaining both the azurin cellular penetration ability and anti-proliferative activity (Yamada et al., 2009).

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

Leu50

Asp77

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

p28 , a peptide formed by aminoacidic residues 50 to 77 of Azurin, shows the same antiproliferative activities of the whole protein.

p28 has been admitted to the Phase II clinical trials under the Food and Drug Administration allowance, but its mode of action has not been completely elucidate yet since the 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 might help to refine the

initial molecule in order to raise its anticancer potentialities

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p28 p28

DBD

p28

NTD

Unbinding force = 95 pN koff = 0.012 ± 0.006 s-1 t = 1/koff = 83 s

Unbinding force= 82 pN koff = 0.13 ± 0.03 s-1 τ = 1/koff = 7.7 s

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. p28 anticancer potentiality could be connected with its ability to hamper the binding of ubiquitin ligase COP1 to p53 DBD.

DBD

Pro DBD TD RD

N1 102 92 64 292 326 353 363 50

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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p53

cop1

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• Elasticity • Viscosity

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It is possible to detect

the elasticity of various

biological samples:

-Cells;

-Bones;

-Collagen;

-Ligament;

-Fat;

-Muscle.

Interest In Bio:

-TRAINING: muscle stiffness;

- OSTEOPOROSIS: lower bone stiffness;

- ARTHROSIS: modification of cartilage

viscoelastic properties;

- VENTRICULAR ANEURYSM: modification of

ventricular viscoelastic properties;

- CANCER: stiffness modification due to cysts.

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Nanomechanical Properties of cell membranes

When the cantilever tip is increasinglypressing on a surface, deflection of thecantilever is lower for the soft samplecompared to the hard sample, due to theelastic bending of the surface and to theindentation of the tip onto the surface.

indentation

The indentantion values are used to extractthe Young’s, or elastic, modulus E of thesample (E measures the elasticity of thematerial).

Indentation is calculated from the difference ofthe cantilever deflection between a soft and a hard surface.

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Hertz model

The hertz model describes a sphere

indenting onto an homogeneous sample

2

3

2

1

213

4δR

v

EF

Stifness

• F and d are read on the indentation curve

• R is estimated

• (u) is estimated

• E can be calculated

)1(4

3 2

2

3

d

FF

RE

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Nanoindentation of viruses

a) The piezo is extending but the AFM tip has not yet touched the virus surface and therefore (b) the exerted force is zero.

c) The AFM tip is indenting

the virus and the cantilever

bends.

d) The change in signal on the quadrant photodiode is a measure for the exerted force, plotted as a function of the indentation.

AFM images of a single viral particle before and after nanoindentation.

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(Sapra et al., 2006)

AFM-forced unfolding of a multidomain Ig-CAM.

Carl et al. PNAS 2001

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AFM-unfolding of Titin. SIF - 20.09.2012

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Further information and literature can be found at the website

http://www.unitus.it/biophysics/ and in our book:

Dynamic Force Spectroscopy and Biomolecular

Recognition

Editor(s): Anna Rita Bizzarri; Salvatore Cannistraro

CRC Press - Taylor & Francis Group

http://www.afm4nanomedbio.eu/home.aspx

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http://www.airc.it/

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THANK YOU FOR YOUR ATTENTION

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Cell and membrane elasticity – calculation of mechanical properties

• Stiff samples: no indentation, straight line

• Soft samples: indentation, curved line

• The indentation curve is given by the difference between the curves on stiff and soft surfaces

To asses the elasticity of the membrane cell, force curves are converted into force vs indentation curves.

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Raw force curves of normal and cancerous cells (human cervical epithelial cells).

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