1

Josef A. KäsInstitute for Soft Matter PhysicsPhysics Department

Scanning Force MicroscopyScanning Force Microscopyi.e. Atomic Force Microscopy

Atomic Force MicroscopyAtomic Force Microscopy

2

Ludwig Maximilians Universität

Hermann E. Gaub Applied Physics

University Munich

Single MoleculeForce Spectroscopy

Zanjan 04

3

4

ForcedLigand-ReceptorUnbinding

Moy, V. T.; Florin, E.-L.; Gaub, H. E. Science 1994, 266, 257-259.

Grandbois, M.; Beyer, M.; Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. Science 1999, 283, p 1727

Keep in mind:

1kBT300K = 25 meV = 4 pNnm = 0.6 kcal/Mol = 2.5 kJ/Mol

Thermal Energy Scale(kBT300K) 1 10 100 1000

Myosinstroke

Molecularrecognition

5

Probing a Single Metallo- Organic Bondthe Ruthenium(II)-Terpyridine Complex

Terpyridine

Functionalized AFM Tip

PEGMetal Ion

SiOx

O - PEG (76) - CH2CH2COOH

N

NN

Ru (II)

80

60

40

20

0Co

unts

5004003002001000Force [pN]

∆G = 40 kT

ProbingMetalloorganicBonds

400

300

200

100

0

-100

-200

For

ce [

pN]

806040200Extension [nm]400

300

200

100

0

-100

-200

For

ce [

pN]

806040200Extension [nm]

8

6

4

2

0

Cou

nts

[%]

100806040200Rupture Length [nm]

6

Unfolding Proteins

Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J. M.; Gaub, H. E. Science 1997, 276, 1109-1112. Oesterhelt, F.; Oesterhelt, D.; Pfeiffer, M.; Engel, A.; Gaub, H. E.; Müller, D. J. Science 2000, 288, 143-146.

The Giant Muscle Protein Titin

Ig module

Fn module

unique insertion sequence

PEVK region

I1 I15 I16 I19 I20 I41

ConstructsI27 I30

Single Ig-Domain

mechanically active part

A-Band I-Band

Z-Disc M-Line

I-Band

Z-Disc

Titin

7

Unfolding 4 and 8 Segment Long Recombinant Titin IgFragments

M. Rief, M. Gautel, F. Oesterhelt, J. M. Fernandez and H. E. Gaub, Science (1997),Vol 276 , p 1109-

250 pN

250 pN

0 50 100 150 200 250

0 50 100 150 200 250

Extension (nm)

Extension (nm)

Fo

rce

Fo

rce

0 50 100 150 200 250

0 50 100 150 200

Extension (nm)

Extension (nm)

600

400

200

0

-200

600

400

200

0

-200

800

Fo

rce

(pN

)F

orc

e (p

N)

50 nm

200 pN

SS

S

1

2

3

200

0

400

0 50 100 150 200 250Extension (nm)

48 74 99 123 147 171 195L(nm)

For

ce (

pN)

p= 3Å

Unfolded Ig 8mer as a Worm Like Chain

F(x)= kTp

1

4(1-x/L)

1

4

x

L( )+-2

8

500

400

300

200

100

0

-100

Forc

e(pN

)

200150100500Extension(nm)

Forced Unbinding of DNA Oligomer Duplexes

PBS,T = 27 C, v = 1.2 µm/s

0.10

0.08

0.06

0.04

0.02

0.00Bon

d ru

ptur

e pr

obab

ility

den

sity

120100806040200Force [pN]

15mer DNA/DNA0.10

0.08

0.06

0.04

0.02

0.00120100806040200

Force [pN]

30mer DNA/DNA

Bon

d ru

ptur

e pr

obab

ility

den

sity

B-S and Melting Transition in l DNA

800

600

400

200

0

Fo

rce

[pN

]

80

60

40

20

0

For

ce [p

N]

4003002001000Extension [nm]

0 1 2

WLC Model p=15 Å

Single Stranded DNA

Double Stranded DNASplit

Strand

Relative Extension

100 pN

Recombination of the Split Strands Reflects Sequence

Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. Nature Struct. Biol. 1999, 6, 346-348.

9

Unzipping DNA Hairpins

Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. Nat. Struct. Biol. 1999, 6, 346-

δ s

δ dCG C

GG

GC

C

Au

100 pN

8006004002000

Extension [nm]

poly dGdC DNA

6004002000Extension [nm]

poly dAdT DNA

FGC

20±3 pN FAT

9±3 pN

25 pN

F

ln (dFpull /dt)

U1

U2

Fpull ⋅⋅ ln

F = kB·T

²x kB·T²x⋅

koff

Energy Landscape Changes under Tension

koff

∆x

∆G

U

x

F > 0

F x

kon

F = 0

xmin =0

kforced

xmax

koff = λ · e kB·T² G-

kforced = λ · e kB·T² G - F·² x-

∆G1∆G2

∆G2 < ∆G1

∆G2 = ∆G1

∆G2 > ∆G1

U

x∆x2

∆x2

R.Merkel and E. Evans., Nature, 397, 50-53

10

Opportunities for Single Molecule Assays in Bio-Analytics

=> Growing demand in Genomics & Proteomics

Current bottle necks:

Sensitivity e.g. Quantitative SNP detection

Selectivity e.g. False positives

High Throughput 106 Assays in parallel

-Mechanics provides orthogonal information-Extremely low amounts of analyte needed

.

.

ImprovedSensitivity by

SmallerSensors

Molecules as Sensors?

Viani, Schäffer, Chand, Rief, Gaub, Hansma J. Appl. Phys. (1999), 4, p2258-

k TB

11

Balances compare unknown forces with a standard

F = k dapple

Scales measure absolute forces

Fapple

?Fstandard

Absolute Versus Differential Force Measurements

Detecting nucleic acid mismatches

Discriminating energetically equivalent binding modes

Singe Molecule Differential Force Assay

12

∆n ≈ ∆I ≈ ∆Pbond

Single Molecule1bit AD-Conversion

Samplebond

Referencebond Parallel Format

∆n ≈ ∆I ≈ ∆Pbond

Single Molecule1bit AD-Conversion

Samplebond

Referencebond Parallel Format

DNA-chip

Elastomer-stamp

Drainage

13

Dis

tanc

e

Force

Cantilever

Dis

tanc

e

Force

Polymer

Deflectionsensor

For

ce

Deflection

Analog signal

For

cePosition

1 bit

Chemically-Programmable Molecular AD Converter

Samplebond

Referencebond

Spots with different reference bonds

14

DNA: a ProgrammableForce Sensor forProtein Chips

- Kerstin Blank- Fillip Oesterhelt

(-> R. Mahaffy et al., PRL, 2000,R. Mahaffy et al., Biophys. J, 86, 1777 (2004) )

AFMAFM--based Measurements of Cell Elasticitybased Measurements of Cell Elasticity

15

AFMAFM--based Measurements of Cell Elasticitybased Measurements of Cell Elasticity

Analyzing Analyzing ViscoelasticViscoelastic AFMAFM--DataDataThe Hertz-model (sphere indenting an infinite elastic half plane):

2/32/1

34 δKRfbead = with K =E/(1-ν2)

viscoelastic extension:tie ωδδδ *

0~

+= δδδ ′′+′= i*~

( )*2/10

*1

2300

2/1 ~23

34 δδδ KKRfbead +≈

*2/10

2/1*1

*

2/30

2/100

~234

δδ

δ

RKf

RKf

osc ≡

≡"'

)(~2 2/10

*

**1 iKK

Rf

K osc +==δδ

with

=>

=>

16

Analyzing Analyzing ViscoelasticViscoelastic AFMAFM--DataData

Correcting for Substrate Correcting for Substrate EffectsEffects (R. Mahaffy et al., Biophys. J, 86, 1777 (2004) )

17

Correcting for Substrate Correcting for Substrate EffectsEffects (R. Mahaffy et al., Biophys. J, 86, 1777 (2004) )

•Tu (for non adhered parts) and Chen model (for adhered parts) correct for substrate effects and simultaneously determine the Poisson ratio.

Elasticity of Elasticity of LamellipodiaLamellipodia

(S.Park et al., Biophys. J, submitted)

18

Elasticity of Elasticity of LamellipodiaLamellipodia

(S.Park et al., Biophys. J, submitted)

2.41.15.8

0.70.385.1

24.712.05.7

12.46.05.7

H-ras (n=10)SEt-test (%)

1.70.63.0

0.60.253.2

12.14.89.6

6.02.49.7

SV-T2 (n=10)SEt-test (%)

0.90.6

0.70.2

7.34.5

3.72.2

BALB (n=10)SE

aDvl30min

Activity(%/min)

DirectionalityMean speed(mm/hr)

Net path length (mm)

0.570.37

0.630.34

1.350.32

Average K (kPa)±

H-rastransformed(n=18)

SV-T2(n=29)

BALB 3T3(n=37)

• Polymerization:1. Thermal ratchet vs. polym.-stick-burst2. Bead motility, nematode sperm motility(-> Formin)

• Molecular motors:1. Knockouts (Spudich, Gerisch) vs. myosin II in the back of

the lamellipodium (Small, Borisy)2. Traction forces on soft substrates (Sheetz, etc)(-> myosin I actin-rail model, cell spreading)

• Retrograde flow:1. Clutch hypothesis

Active Force Generation IActive Force Generation I

19

Active Force Generation IIActive Force Generation II

(-> C. Brunner, M. Gögler, A. Ehrlicher, T. Jähnke, B. Kohlstrunk, Propulsive forces of fast moving cells, Nature, in preparation (2004) )

Active Force Generation IIActive Force Generation II

(-> C. Brunner, M. Gögler, A. Ehrlicher, T. Jähnke, B. Kohlstrunk, Propulsive forces of fast moving cells, Nature, in preparation (2004) )

Translocation [µm]

Def

lect

ion

[nm

]

lamellipodiumcell body

20

Active Force Generation IIIActive Force Generation III

Active Force Generation IIIActive Force Generation III