University of Linz

Austria

Single Molecule Recognition

Atomic Force Microscopy

Peter Hinterdorfer

Purdue
10-05-2006

1
 Overview

¾ MACmode AFM Imaging

¾ Recognition Force Spectroscopy

¾ Combining Force Spectroscopy with Imaging

¾ Simultaneously recorded Topography and

Recognition images (TREC)

¾ Summary and Outlook

2
 MACmode AFM

3
 AFM Tips and Cantilevers

http://www.park.com http://www.park.com

normal tip EBD tip

3 µm tall, ~30 nm end radius

4 http://stm2.nrl.navy.mil/how-afm/how-afm.html#tips
 MACmode Imaging

Amplitude-distance cycle using a bare AFM-tip

14

12

10
Amplitude [nm]

6 freeamplitude
amplitudereduction
Setpoint value
4

0 imaging amplitude
-2
-60 -40 -20 0 20 40 60 80

Distance [nm]

5
 Force Detection

6
 Tip Chemistry (via flexible linker)

1 PEG

HN O

O
S
N S S
PEG
H
Acetyl N 2

HN O

NH2
3
O H
+ NaCNBH3 PEG

BIOTIN

7 Riener et al., Anal. Chim. Act. 497 (2003) 1367

 SH- or His6-coupling
I. tip-PEG-PDP + HS-biomolecule II. tip-SH + PDP-PEG-NTA + His6-protein

O 1
1
NH2 O N SH
H S N
S
O O N

PEG O
O NH
HN O
PEG

S N O
N S 2 H
O HOOC N COOH
HS N Ni2+ COOH
H His6
3 2
2 VLDLR1-3

1
NH2
8 Riener et al., Anal. Chim. Act. 497 (2003) 1367
 Direct NH2-Coupling
III. tip-PEG-aldehyde + NH2-protein IV. tip-PEG-vinylsulfon + NH2-protein

1 1
NH2 O NH2 O

O O N O O N

PEG O PEG O

HN O HN O

H2N O S O
H O
H2N
2
2

9 Riener et al., Anal. Chim. Act. 497 (2003) 1367

 Surface Chemistry

1 PEG

HN O

O
S
N S S
PEG
H
A cetyl N 2

HN O

N H2
3
O H
+ N aC N BH 3 PEG

STREPTAVIDIN B IO T IN

NH2

glutaric aldehyde

A H2N B

APTES AVIDIN
O Si O
O

10 Riener et al., Anal. Chim. Act. 497 (2003) 59

 Force-Distance Cycle
cantilever

tip

antibody

lysozyme

mica

11 Hinterdorfer et al., PNAS 93 (1996) 3477, Nanobiol. 4 (1998) 177

 Probability Density

12
 Probability Density

13
 Probability Density

14
 Theory of Force-Spectroscopy

Master Equation Boltzmann Ansatz

dN(t)/dt = -kd(rt)N(t) kd(F) = kd(0) eFx/kT

F*(r) = kBT/xß ln(r xß/ koff kBT)

• • •

E. Evans, T. Strunz
15
 Molecular Complexes & Forces
150
150
150 nm
Cantilever 150nm
150 nm
nm
nm 72
0072
48 min
min
48
minmin
min
min
NH2
NH2
300 NH2
NH2

NHS
200
Force [pN]

100

2 Fab’s of
PEG
0
antibody
-100

PDP
-200
SATP
0 5 10 15 20 25 30

Extension
Antibody [nm] (I)
3 nm
400
24 aa 101 aa 161 aa 232 aa
(II)
300 BR-trimer 0 nm
Force [pN]

(III)
200

100

0
Fab (IV)
Fc
0 20 40 60 80 100
Fab
Extension [nm]
30 nm

16Kienberger et al., J. Mol. Biol. 347 (2005) 597-606 Kienberger et al., EMBO Rep. 5 (2004) 579-583
 Human Rhinovirus

VLDLR1-8 VLDLR1-3

Extracellular Side
Plasmamembrane
Intracellular Side

+
17
Empty Capsid RNA
 Substructure of RNA
60 nm 80 nm

18 Kienberger et al., J. Virol. 78 (2004) 3203-9

 Virus-Receptor Complex
10 nm
25 nm

0 nm

25 nm
25 nm 4
5
10 nm 3
2
1
0 nm
5
4
3
2
1
20 nm

19Kienberger et al., Structure, in press Kienberger et al., J. Virology 78 (2004) 3203-9

 Virus-Receptor Interaction

Cantilever
SATP
SATP
SATP
SATP

(III) PDP

+ Ca2+ - Ca2+
(II) PEG

(I) NTA
His6

25 nm VLDLR

20 Rankl et al., manuscript in preparation

 Receptor Constructs against HRV2

Cantilever
SATP
SATP
SATP
SATP

PDP

PEG

NTA
His6

VLDLR

21 Rankl et al., manuscript in preparation

22
fit linear function f=k*ln r + d
Rankl et al., manuscript in preparation
 k = 1 / xβ

dÎkoff

23 Rankl et al., manuscript in preparation

24 Rankl et al., manuscript in preparation
25 Rankl et al., manuscript in preparation
26 Rankl et al., manuscript in preparation
 Results: VLDLR-HRV2 Binding

construct xβ [nm ] koff [s-1]

V1-8 0,41± 0,18 0,088± 0.067
V1-3 0,79± 0,24 0,11± 0.083
V333 0,37± 0.037 0,75± 0,58
V33 0,62± 0,12 0,83± 0,76

Xβ [nm] koff [s-1]

0,8 V1-3 0,8 V33,V333
0,5
0,6 V33
0,4 V1-8, V333 0,1 V1-8,V1-3
27 Rankl et al., manuscript in preparation
 Lateral Force Mapping

Recognition Map Block Binding Probability

0.015

Pdf (1/pN)
0.01

0.005

0
0 100 200 300

Force (pN)

50 nm 50 nm

64 X 64 Pixels TExp = 14 min

28 Stroh et al., Biophys. J. 87 (2004) 1981

 Principles of TREC
Topography Recognition

Recognition:
A Amplitude Reduction
on Top

Topography:
Amplitude Reduction
on Bottom
29
 MacMode Force Traces
Envelope of cantilever oscillation

Bare tip

Single molecules on mica

Slow scan axis disabled

Coated tip
Slow scan axis enabled

150 nm

30 Stroh et al., Biophys. J. 87 (2004) 1981

 Repeated Linear Scans
Bottoms Tops of Amplitudes

Bare Tip

Antibody Tip

1 nm Block
0 nm
200 nm

31 Stroh et al., Biophys. J. 87 (2004) 1981

 TREC Scheme
TREC = Simultaneous Topography and RECognition Imaging

Recognition
Image
Uup

MAC TREC
BOX BOX AFM
CONTROLLER
Udown
feedback loop

Topography
Image

32 Stroh et al., Biophys. J. 87 (2004) 1981

 Topography & Recognition
512 x 512 Pixels TExp = 8 min

Topography image of Simultaneously acquired

avidin adsorbed on mica recognition image

150 nm 150 nm

33 Ebner et al., ChemPhysChem, 6 (2005), 897

 Application to Chromatin
Topography Recognition

50nm
mmtv chromatin/anti-histone H3 on tip

34 Stroh, Wang, et al., PNAS 101 (2004) 12503

 Recognition is specific
Anti Histone H3 on
non-acetylated
MMTV

+30μg/ml BSA
• No blocking

+ 50μg/ml
ARTKQTARKSTGGKAPRKQLC
(aa 1-20 of H3)

35 Stroh, Wang, et al., PNAS 101 (2004) 12503

 Accuracy and Repeatability

Green = ‘hit’ first scan

96±1% (false1.1%±0.1%)
Blue = ‘miss’ second scan
Red = ‘false hit’ 92±2%, (false 2.8%±0.5)
Arrows = change on rescan

36 Stroh, Wang, et al., PNAS 101 (2004) 12503

 Distribution of trans-interacting VE-cadherins
on MyEnd surface
Microvascular endothelial cell line from mouse myocardium (MyEnd)
Vascular endothelial cadherin (calcium-dependent adherent protein) (VE-cadherin)
Selective adhesion between cells Immunofluorescence labeling of VE-cadherin

(Baumgartner W et al., Histochem Cell Biol, 2004)

Single molecule fluorescence imaging

5 × 103
cis dimers
μm 2
- Lateral resolution is not better
than 200 nm
- No information about
topography (Baumgartner W et al, J Cell Sci, 2003)
37
 AFM Simultaneous Topography and RECognition (TREC) TECHNIQUE
MAC Mode
TREC scheme
- Good for “soft” samples
(proteins, cells, etc.)
- Gentle imaging technique
- Physiological environment
Æ Requirement for TREC

topography recognition
model system: avidin-biotin

AFM tip:
biotin via PEG linker
mica surface:
avidin molecules

blocking with
streptavidin

38 (Stroh C. et al. BiophysJ 2004, PNAS 2004; Ebner A. et al. ChemPhysChem,2005)

 Morphology of MyEnd cells single live cell
AFM PicoPlus; Large scanner; Cantilever: tip E (ν≈7,5 kHz); HBSS at RT
Contact mode Mac mode

20 μm 20 μm

Lateral mobility of receptors on cell membrane

Fixation of cells!
39
 Morphology of MyEnd cells fixed cells
fixation with glutaraldehyde

normal fixation in buffer gentle fixation in medium

(Oberleithner H. et al. Hypertension, 2004)

15 μm 5 μm

Filamentous network at the cell cortex is

mostly conserved!
40
 Topography of gently fixed MyEnd cells
cytoskeleton organisation
AFM-MAC mode Transmission EM

F-actin filaments

microtubules

(Heuser and Kirschner, JCB, 1980)

41
 TREC on fixed MyEnd cells / VE-cadherin
AFM tip (ν~7.5 kHz): scan size: 1.7μm x 1.7μm
VE-cadherin-Fc via PEG-linker scan speed: ~ 3 μm/s
recognition map blocking with 5mM EDTA

specific binding site

or not?

300 nm 300 nm

42
 TREC on fixed MyEnd cells / VE-cadherin
recognition topography

size of VE-cadherin’s
microdomains
from ~ 30 nm
to ~ 500 nm

+EDTA

43 300 nm 300 nm
 Force measurements: VE-cadherin cis-
dimers interaction
AFM tip:
VE-cadherin-Fc
via PEG-linker

MyEnd surface VE-cadherin cis-dimers on mica

μ1

μ2 μ3

(Baumgartner W et al, PNAS, 2000)

44
 TREC on fixed MyEnd cells / Fibrinogen
Specificity of fibrin(ogen) to endothelial cells
-VE-cadherin
-Integrin αVβ3
-Intercellular adhesion molecule (ICAM-1)

topography recognition force distribution

200 nm 200 nm

45
 Summary

¾MacMode Imaging under Physiological Conditions

¾Single Molecule Recognition Force Spectroscopy

¾Simultaneous Mapping of Topography and Molecular Recognition

¾ Nanometer Lateral Resolution at fast Acquisition Rates

46
 People involved

AFM-group University of Linz Technical support

Ferry Kienberger Manfred Geretschläger

Christian Rankl Günther Freudenthaler
Cordula Stroh
Lilia Chtcheglova
Andreas Ebner
Theeraporn Puntheeranurak
Rong Zhu

Surface Chemistry: Hermann Gruber, Linda Wildling,

Christoph Hahn, Christian Riener
47
 People involved

Reinat Nevo, Ziv Reich (Weizmann Institute)

Rosita Moser, Dieter Blaas (Vienna Biocenter)
Werner Baumgartner (University of Aachen)
Harald Müller (University of Kassel)
Sandra Smith-Gill (NIH)
Carina Huber; Margit Sara (BOKU Vienna)
Gerald Pfister, Georg Wick (OEAW, Innsbruck)

Arizona State University Molecular Imaging, Tempe

Hongda Wang, Brian Ashcroft, Gerald Kada, Travis Johnson,
Stuart Lindsay Jeremy Nelson, Tianwei Jing

48
 http://www.molec.com/linz2007.html

Biological single-molecule research, nano-science, nano-medicine, bio-nanotechnology

Techniques:

• atomic force microscopy

• dynamic force spectroscopy
• optical tweezers
• nanofabrication methods
• self-organization
• fluorescence microscopy
• optical spectroscopy
49
 University of Linz
Austria

Peter Hinterdorfer

Purdue
10-05-2006

50