Applicazioni della microscopia a forza atomicaper lo studio di DNA

Corso di perfezionamento in TECNICHE DI MICROSCOPIA A FORZA ATOMICA - Parma 29 febbraio 2012

Prof. Claudio RivettiDipartimento di Biochimica e Biologia MolecolareUniversità degli Studi di Parma

Gro-EL and Gro-ES chaperonins

φ29 head-tail connector

SupercoilingPersistence lengthIntrinsic bendingLabeling sequences and structures

Binding site localizationProtein stoichiometryDNA bendingDNA loopingDNA wrappingSubunit localization

Transcription

Protein conformational changes

AFM Applications in Biology

SupercoilingPersistence lengthIntrinsic bendingLabeling sequences and structures

Binding site localizationProtein stoichiometryDNA bendingDNA loopingDNA wrappingSubunit localization

Protein structure from 2D crystals

Real-time visualization of biochemical processes

Protein Folding

DNA structure

Protein-DNA Complexes

Struttura del DNA

3.4 nm

= 2 nm

TBP Topoisomerase I

In a crystalline Solid

A piece of household Aluminium foil

4 mm

∆x

F

F

1 mm

∆x

X0

F FF = k∆x

k ~ 10 N/m

F = k∆x

k ~ 1 N/m

laser

mirror

cantilever

piezo

Atomic Force Microscope

photodiode

mica

Contact mode AFM

Tapping mode AFM

scan

scan

Tip

The broadening effect of the scanning tip

Scan direction

Spatial resolution in SFM depends on tip dimensions

Tip

2Rm

Rc

R’c

W

W’

4 cm RRW =

Una proteina globulare con diametro 5 nm visualizzata mediante una punta con un Rc di 5nm, appariràcon una dimensione di 20 nm

Spatial Resolution in SFM

( )d R z z h d R hc c= + + >2 2∆ ∆ ∆ ∆ for

Inverted TipSurfaces

Rc

∆z

d

TipTip

A B

∆z∆h

SampleSpikes d

Scan direction

Rc

Pyramidal Silicon Nitride SFM Tip

4 nm

Pyramidal silicon nitride tips Electron beam-deposited tip

Carbon nanotube tip

Different biological samples Different biological samples

200 nm 200 nm

15.4 nm

9.3 nm0 nm

2 nm

Regular tip Tip with nanotube

Tip

Rc

Rc = 1.0 nm Rc = 0.5 nm Rc = 0.1 nm

Rc = 7.0 nm Rc = 5.0 nm Rc = 4.0 nm Rc = 2.0 nm

Rc = 0.1 nm

20 nm

3.0 nm

AFM image simulation of DNA

MICA

DNA deposition methods

Polycations(spermidine, polyornithine)

DNA adhesion on mica

Limiti dimensionali del DNA

DNA: da 200 bp a 2000 bp 220 bp 223 bp

800 bp 1500 bp

DNA fago λ : 48500 bp

RNA e DNA a singolo filamento?

1 µm

Limiti dimensionali delle proteine

Proteine: da 20 KDa a 1000 kDa

Sak monomer 20 kDa

2 µm 3 µm

Batterio

Quantità di materiale richiesto

Generalmente con 10 ng di DNA si ottiene una buona deposizione su di un supporto di 1 cm2

Teoricamente si potrebbero fare 25 milioni di immagini 2x2 um.

2 µm

Quali complessi DNA/proteina si possono studiare?

Per avere metà del DNA legato dalla proteina devo utilizzare una concentrazione di proteina almeno uguale alla Kd del complesso.

Negli esperimenti in soluzione generalmente la concentrazione di proteina non è quasi mai limitante.

In AFM invece, un eccesso di proteina libera satura la mica al punto che il DNA ed i complessi non riescono più a depositarsi.

Il limite dipende dalle dimensioni della proteina ma generalmente, complessi con Kd > 100 nM sono difficili da visualizzare.

Complessi formati da più proteine?

1 2

3 4

Influence of the mica surface on protein stability

Sak Sak + Glutaraldheide 0.11%

How do DNA molecules go fromsolution to the surface?

Once bound to the surface can they return into solution?

What happens to the molecules on the surface before removing the buffer?Can they move in 2D or are theytrapped in a single conformation?

Can we quantitatively distinguishbetween the different cases?

Imaging DNA molecules

DNA deposition steps

DNA Deposition onto Mica

0 nm

4 nmFreshly cleaved Glow discharged H+-exchanged

2 µm 2 µm 2 µm

Number of DNA Molecules on the Surface vs. Time

Time (s)

1 10 100 1000n

F/n

0(1

/cm

)1

10

10010-4

slope = 0.49±0.04

Time (s)

0 1000 2000

nF

/n0

0.0

0.2

0.4

0.6

Mo

lec

ule

sin

4x4

µm

2

0

192

384

576

D = (5.4±0.2)10-8 cm2/s

If transfer of DNA molecules from solution onto the surface is solely governed by diffusion:

nF

n0

4D= tπ

nF is the number of molecules on the surfacen0 is the total number of molecules in solutionD Diffusion coefficientt timeThis equation is valid if:

• The molecules are irreversibly adsorbed to the surface• Convection currents do not contribute to the transport of the molecules to the surface• The solution is not significantly depleted of DNA molecules during the time of deposition• The surface is not saturated during the time of deposition

DNA DNA imagedimaged in in liquidliquid

Equilibrium Statistic of a Worm-like Chain

In 2D the mean square end-to-end distance of a worm-like chain of length L, and persistence length P, is:

R PL PL

eD

LP2

224 1 2 1= − −

⎛

⎝⎜⎜

⎞

⎠⎟⎟

⎛

⎝⎜⎜

⎞

⎠⎟⎟

−For L R PL

D→∞ =2

24

L

Ru1

u2

The persistence length of the molecule, P,is the decay length through which the initialorientation of the molecule persists. It is ameasure of the stiffness of a polymer chain.

r ru u eLP

1 2⋅ =−

DNA contour length (nm)

0 500 1000 1500 20000

1

2

3

4

5

6

0 250 5000

.5

1

105

105

<R

2>

(n

m2)

4 mM Hepes pH 7.4, 10 mM NaCl, 2 mM MgCl24 mM Hepes pH 7.4, 10 mM NaCl, 100 mM MgCl210 mM Hepes pH 8.0, 80 mM NaCl, 5 mM MgCl210 mM Hepes pH 6.8-8.0, 5 mM NaCl, 5 mM MgCl2

P = 53 nm

100 nm

1258 bp DNA

Theoretical model for a 1258 bp DNA <R2> nm2

Ideal worm-like chain in 3D 35600Ideal worm-like chain in 2D 60500Orthogonal 3D → 2D projection 23700

Freshly cleaved Glow discharged H+-exchanged

<R2> = 61300 nm2 <R2> = 26000 nm2 <R2> = 25100 nm2

DNA bend angle measurements

Tip

Hidden DNA

β

β

RR

AAAAAACGCGTTTTTTGCGC

AAAAAACGCGTTTTTTGCGC

10 bpA-tract

AAAAAACGCGTTTTTTGCGC

AAAAAACGCGTTTTTTGCGC

Using the end-to-end Distance to Determine Bend Angles

For a polymer molecule that is bentat any location along the chain, themean square end-to-end distanceis given by:

β

R

L-ll

( ) ( )( ) ( ) ( )( )[ ]⎭⎬⎫

⎩⎨⎧ −−−−+−−= −−−−−− PLPPLP

Deeee

LPPLR 2222

2

2 11)cos(11214 llll ββ

2 µm50 nm

2 A-tracts

8 A-tracts7 A-tracts6 A-tracts

5 A-tracts4 A-tracts3 A-tracts

2 A-tracts

8 A-tracts

6 A-tracts4 A-tracts

DNA fragments containing A-tracts

Mean square end-to-end distance as a function of the number of A-tracts

Number of A-tracts0 2 4 6 8 10

<R2 >

(nm

2 )

7.0

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0103

[AAAAAACGCG]155 bp 206 bp

n=1-8

T=25 °C

β per A-tract = 14 deg.

<R2> of polymers made of regions with different persistence length

For a polymer molecule made of threeregions with different flexibility, the meansquare end-to-end distance is given by:

( ) ( ) ( )

( )( ) ( )( ) ( )( )331133222211

332211

118118118

114114114

313221

3

333

2

222

1

1112

2

PPPPPP

PPP

D

eePPeePPeePP

ePPePPePPR

llllll

lll

ll

ll

ll

−−−−−−

−−−

−−+−−+−−+

+⎥⎦

⎤⎢⎣

⎡−−+⎥

⎦

⎤⎢⎣

⎡−−+⎥

⎦

⎤⎢⎣

⎡−−=

rR

l 1, P 1

l2, P2

l3, P3

50 nm

0 T (dsDNA)

5 T

3 T1 T

10 T

DNA fragments containing ssDNA gaps

2 µm

Number of T in the ssGAP0 5 10 15 20 25

12

14

16

18

20

22103

<R2 >

(nm

2 )

Mean square end-to-end distance as a function of the numberof T in the ssDNA gap

[ Tss ]299 bp 334 bp

n

PssDNA = 1.2 nm

Structure of the E. coli RNA Polymerase

Template A 1008 bp

Template C 1150 bp

λPR439 bp 569 bp

+1

λPR439 bp 413 bp

λPR298 bp

+1 +1

AFM image of Open Promoter Complexes

280 nm

325 nm

289 nm

306 nm

363 nm295 nm351 nm

363 nm

323 nm

174 nm

Pol

10 nm

30 nm

36 nm

DNA

6 nm of DNA compaction

βR

DNA Contour Length Measurements of Open Promoter Complexes

200 250 300 350 4000

30

60

90

120

Contour Length (nm)

DNAOPC

240 260 280 300 320 340 360 380 4000

10

20

30

40

50

60DNA1 OPC2 OPC

Contour Length (nm)

Num

bero

f mol

ecul

es

Num

bero

f mol

ecul

esOPC on Template A OPC on Template C (2 λPR)

DNA (A) 329 ± 12 947OPC (A) 297 ± 34 32 514

DNA (C) 363 ± 8 317One OPC (C) 332 ±14 31 157Two OPC (C) 308 ± 20 55 173

Contour length (nm) Compaction (nm) N. of molecules

P~100 Å

~60°

~300°

+1

Downstream DNA

UpstreamDNA

+24

-70

Upstream DNA

Downstream DNA

+24

-70

-38 +1

Proposed model for the open promoter complex at λPR

200 nm

λPR

λPR

λPR

λPR

λPR

-100

-79

-59

-35

wt

PR -100Wt PR PR -79 PR -59 PR -35

0

25

50

300 310 320 330 340 350

Freq

uenc

y

0

25

0

25

50

270 280 290 300 310 320 330

Freq

uenc

y

0

20

0

15

30

270 280 290 300 310 320 330

Freq

uenc

y

0

12

0

18

36

270 280 290 300 310 320 330

Freq

uenc

y

0

22

0

20

40

270 280 290 300 310 320 330

Freq

uenc

y

0

20

D

CBA

E

30 ±0.3 nm 30 ±0.4 nm 20 ±0.5 nm

20 ±0.5 nm 4 ±0.7 nm

wt PR – wt PRM PR (-100) PR (-79)

PR (-59) PR (-35)

Contour length (nm)

Contour length (nm) Contour length (nm) Contour length (nm)

Contour length (nm)

OPC with mutant λPR promoters

-20-30-40

-50

-60

-70

-80

-90

-100

-10

+20

+30PRM

PR

-20-30-40

-50

-60

-70

-80

-90

-100

-10

+20

+30

PRM

PR

-20-30-40

-50-60

-70-80

-90

-100

-10

+20

+30

PRM

PR

325 bp372 bp

445 bp 502 bp

tab3 tab2 tab1

TraA(70 kDa)

TraA-cPD1

TraA-iPD1

100 nm

Binding of TraA to tab1, tab2 and tab3

0.20 0.25 0.30 0.35 0.40 0.45 0.500

10

20

30

40

50

0.20 0.25 0.30 0.35 0.40 0.45 0.500

5

10

15

20

25

30

0.20 0.25 0.30 0.35 0.40 0.45 0.500

10

20

30

40

Freq

uenc

y

TraA position TraA position TraA position

tab3 tab2 tab1 tab3 tab2 tab1 tab3 tab2 tab1

325 bp372 bp

445 bp 502 bp

tab3 tab2 tab1

TraA position along a DNA fragment containing tab1, tab2 and tab3

tab3 tab2 tab1

0.26 0.17 0.57

tab3 tab2 tab1

0.45 0.23 0.32

tab3 tab2 tab1

0.26 0.22 0.52

TraA TraA-cPD1 TraA-iPD1

Freq

uenc

y

RNAP position

PA+P0

+

533 bp 131 243 bp

0.1 0.2 0.3 0.4 0.50

10

20

30

RNAP position

533 bp 131 243 bp

PA+P0

-

0.1 0.2 0.3 0.4 0.50

15

30

45

60

RNAP position

533 bp 131 243 bp

P0+ PA

-

0.1 0.2 0.3 0.4 0.50

10

20

30

40

50

Reassessing the PA position by AFM

Antibody

Z-DNA

B-DNA

Avidin

Triple Helix

B-DNA

Labeling DNA Sequences and DNA Structures Labeling DNA Sequences and DNA Structures

LabelingLabeling the the ββ’’ subunitsubunit of of an RNAPan RNAP

100 nm

β’RNAP

DNA tag

Streptavidin

Biotin

Upstream DNA Downstream DNA

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Rivetti C, Walker C, Bustamante C (1998). Polymer Chain Statistics and Conformational Analysis of DNA Molecules with Bends or Sections of Different Flexibility. J. Mol. Biol. 280 41-59.

Rivetti C, Guthold M, Bustamante C (1999). Wrapping of DNA around the E. coli RNA polymerase open promoter complex. EMBO J. 18 4464-4475.

Cellai S, Mangiarotti L, Vannini N, Naryshkin N, Kortkhonjia E, Ebright RH, Rivetti C (2007). Upstream promoter sequences and alphaCTD mediate stable DNA wrapping within the RNA polymerase-promoter open complex. EMBO Rep 8 271–278.

Mangiarotti L, Cellai S, Ross W, Bustamante C, Rivetti C (2008). Sequence-Dependent Upstream DNA-RNA Polymerase Interactions in the Open Complex with lambdaP(R) and lambdaP(RM) Promoters and Implications for the Mechanism of Promoter Interference. J Mol Biol 385 748–760.

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Hamon Loic, David Pastré, Pauline Dupaigne, Cyrille Le Breton, Eric Le Cam and Olivier Piétrement. (2007). High-resolution AFM imaging of single-stranded DNA-binding (SSB) protein-DNA complexes. Nucleic Acids Research, 35:8 e58.

Lyubchenko, Y.L., Shlyakhtenko, L.S., Harrington, R.E., Oden, P.I. and Lindsay, S.M. (1993), “Atomic force microscopicy of long DNA : imaging in air and under water” Proc. Natl. Acad. Sci. USA, 90, 2137-2140.

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References