Atomic Force Microscopy - uni-leipzig.de · 2007-01-12 · Force [pN] 15mer DNA/DNA 0.10 0.08 0.06...
Transcript of Atomic Force Microscopy - uni-leipzig.de · 2007-01-12 · Force [pN] 15mer DNA/DNA 0.10 0.08 0.06...
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
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