single molecule recognition atomic force microscopy

University of LinzAustria

Single Molecule Recognition Atomic Force MicroscopyPeter HinterdorferPurdue 10-05-2006 1

OverviewMACmode AFM Imaging Recognition Force Spectroscopy Combining Force Spectroscopy with Imaging Simultaneously recorded Topography and Recognition images (TREC) Summary and Outlook2

MACmode AFM

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AFM Tips and Cantilevers

http://www.park.com

http://www.park.com

normal tip

EBD tip

3 m tall, ~30 nm end radius

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

MACmode ImagingAmplitude-distance cycle using a bare AFM-tip14 12

Amplitude [nm]

10 8 6 4 2 0 -2 -60 -40 -20 0 20 40 60 80

free amplitudereduction amplitude Setpoint value

imaging amplitude Distance [nm]

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Force Detection

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Tip Chemistry (via flexible linker)

1

PEG

HN O N H S Acetyl S S

O

N

2

PEG

HN

O

NH2

O + NaCNBH3

H

3PEG

BIOTIN

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

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

NH2 O

1

O

1 SH S O S N

O O N

N H

PEG HN O

O

NH PEG

N

S

S

2

O N H3 2 1

HOOC

N

N O H COOH COOH

HS

His6VLDLR1-3

Ni2+

2

8

NH2


Direct NH2-CouplingIII. tip-PEG-aldehyde + NH2-protein IV. tip-PEG-vinylsulfon + NH2-protein

NH2 O

1

O O N

NH2 O

1

O O N

PEG HN O

O

PEG HN O

O

H

O 2

H2N

O S O H2N 2

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Surface Chemistry1PEG

HN O N H S A cetyl S S

O

N

2

PEG

HN

O

N H2

O + N aC N BH 3

H

3PEG

STREPTAVIDINNH2

B IO T IN

glutaric aldehyde

A

H2N

BAPTES AVIDIN

O Si O O

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Force-Distance Cyclecantilever tip antibody

lysozyme

mica

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

Probability Density

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Probability Density

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Probability Density

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Theory of Force-SpectroscopyMaster EquationdN(t)/dt = -kd(rt)N(t)

Boltzmann Ansatzkd(F) = kd(0) eFx/kT

F*(r) = kBT/x ln(r x/ koff kBT)

E. Evans, T. Strunz

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Molecular Complexes & ForcesCantileverNH2 NH2 NH2

150 nm 150 nm 150 nm 150 nm 150 nm

72 min 048min min 048 min min 72 min

300

NH2

NHS200

Force [pN]

100

0

PEG

2 Fabs of antibody

-100

-200

PDP0 5SATP 10

15

20

25

30

Extension [nm] Antibody

(I)3 nm232 aa

400 24 aa 300 101 aa 161 aa

BR-trimer

0 nm

(II) (III)Fab Fc

Force [pN]

200

100

0 0 20 40 60 80 100

(IV)

Extension [nm]

Fab

30 nm

16 Kienberger et al., J. Mol. Biol. 347 (2005) 597-606

Kienberger et al., EMBO Rep. 5 (2004) 579-583

Human Rhinovirus

VLDLR1-8

VLDLR1-3Extracellular SidePlasmamembrane

Intracellular Side

+17

Empty Capsid

RNA

Substructure of RNA60 nm

80 nm

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

Virus-Receptor Complex25 nm10 nm 0 nm

25 nm25 nm10 nm

5 4 3 2 1

0 nm

20 nm

5 4 3 2 1

Kienberger et al., Structure, in press 19

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

Virus-Receptor InteractionCantileverSATP SATP SATP SATP

(III)

PDP

(II)

PEG

+ Ca2+

- Ca2+

(I)25 nm

NTAHis6

VLDLR

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Rankl et al., manuscript in preparation

Receptor Constructs against HRV2CantileverSATP SATP SATP SATP

PDP

PEG

NTAHis6

VLDLR

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fit linear function f=k*ln r + d


k = 1 / x

d

koff

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24


25


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Results: VLDLR-HRV2 Bindingconstruct V1-8 V1-3 V333 V33 x [nm ] 0,41 0,18 0,79 0,24 0,37 0.037 0,62 0,12 koff [s-1] 0,088 0.067 0,11 0.083 0,75 0,58 0,83 0,76

X [nm]0,8 0,6 0,427

koff [s-1]V1-3 V33 V1-8, V3330,8 0,5 0,1

V33,V333

V1-8,V1-3


Lateral Force MappingRecognition Map Block Binding Probability0.015

df P (1/pN)

0.01

0.005

0

0

100

200

300

F orce (pN)50 nm 50 nm

64 X 64 Pixels28

TExp = 14 minStroh et al., Biophys. J. 87 (2004) 1981

Principles of TRECTopography Recognition

Recognition: A Amplitude Reduction on Top Topography: Amplitude Reduction on Bottom29

MacMode Force TracesEnvelope of cantilever oscillation Bare tip Single molecules on micaSlow scan axis disabled

Coated tipSlow scan axis enabled

150 nm

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

Repeated Linear ScansBottoms Tops of Amplitudes Bare Tip

Antibody Tip

1 nm 0 nm

Block200 nm

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TREC SchemeTREC = Simultaneous Topography and RECognition ImagingRecognition Image Uup MAC BOX TREC BOX Udown AFM CONTROLLER feedback loop Topography Image

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Topography & Recognition512 x 512 Pixels Topography image of avidin adsorbed on mica TExp = 8 min Simultaneously acquired recognition image

150 nm33

150 nmEbner et al., ChemPhysChem, 6 (2005), 897

Application to ChromatinTopography Recognition

50nmmmtv chromatin/anti-histone H3 on tip

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

Recognition is specificAnti Histone H3 on non-acetylated MMTV

+30g/ml BSA No blocking

+ 50g/mlARTKQTARKSTGGKAPRKQLC (aa 1-20 of H3)35 Stroh, Wang, et al., PNAS 101 (2004) 12503

Accuracy and Repeatability

Green = hit Blue = miss Red = false hit Arrows = change on rescan36

first scan 961% (false1.1%0.1%) second scan 922%, (false 2.8%0.5)

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

Distribution of trans-interacting VE-cadherins on MyEnd surfaceMicrovascular 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 m 2- Lateral resolution is not better than 200 nm - No information about topography

cis dimers

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(Baumgartner W et al, J Cell Sci, 2003)

AFM Simultaneous Topography and RECognition (TREC) TECHNIQUEMAC Mode- Good for soft samples (proteins, cells, etc.) - Gentle imaging technique - Physiological environment Requirement for TREC

TREC scheme

topography

recognition

model system: avidin-biotinAFM tip: biotin via PEG linker mica surface: avidin molecules

blocking with streptavidin

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

Morphology of MyEnd cellsContact mode Mac mode

single live cell

AFM PicoPlus; Large scanner; Cantilever: tip E (7,5 kHz); HBSS at RT

20 m

20 m

Lateral mobility of receptors on cell membrane

Fixation of cells!39

Morphology of MyEnd cellsfixation with glutaraldehydenormal fixation in buffer

fixed cells

gentle fixation in medium(Oberleithner H. et al. Hypertension, 2004)

15 m

5 m

Filamentous network at the cell cortex is mostly conserved!40

Topography of gently fixed MyEnd cellsAFM-MAC mode Transmission EM

cytoskeleton organisation

F-actin filaments

microtubules

(Heuser and Kirschner, JCB, 1980)

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TREC on fixed MyEnd cells / VE-cadherinAFM tip (~7.5 kHz):VE-cadherin-Fc via PEG-linker scan size: 1.7m x 1.7m scan speed: ~ 3 m/s

recognition map

blocking with 5mM EDTA

specific binding site or not?

300 nm

300 nm

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TREC on fixed MyEnd cells / VE-cadherinrecognition topography

size of VE-cadherins microdomains from ~ 30 nm to ~ 500 nm

+EDTA

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300 nm

300 nm

Force measurements: VE-cadherin cisdimers interactionAFM tip: VE-cadherin-Fc via PEG-linker MyEnd surface 1 VE-cadherin cis-dimers on mica

2

3

(Baumgartner W et al, PNAS, 2000)

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TREC on fixed MyEnd cells / FibrinogenSpecificity of fibrin(ogen) to endothelial cells-VE-cadherin -Integrin V3 -Intercellular adhesion molecule (ICAM-1)

topography

recognition

force distribution

200 nm

200 nm

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Summary

MacMode Imaging under Physiological Conditions Single Molecule Recognition Force Spectroscopy Simultaneous Mapping of Topography and Molecular Recognition Nanometer Lateral Resolution at fast Acquisition Rates

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People involvedAFM-group

University of Linz

Technical support Manfred Geretschlger Gnther Freudenthaler

Ferry Kienberger Christian Rankl Cordula Stroh Lilia Chtcheglova Andreas Ebner Theeraporn Puntheeranurak Rong Zhu

Surface Chemistry: Hermann Gruber, Linda Wildling, Christoph Hahn, Christian Riener47

People involvedReinat Nevo, Ziv Reich (Weizmann Institute) Rosita Moser, Dieter Blaas (Vienna Biocenter) Werner Baumgartner (University of Aachen) Harald Mller (University of Kassel) Sandra Smith-Gill (NIH) Carina Huber; Margit Sara (BOKU Vienna) Gerald Pfister, Georg Wick (OEAW, Innsbruck) Arizona State University Hongda Wang, Brian Ashcroft, Stuart Lindsay48

Molecular Imaging, Tempe Gerald Kada, Travis Johnson, Jeremy Nelson, Tianwei Jing

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

Biological single-molecule research, nano-science, nano-medicine, bio-nanotechnologyTechniques:

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atomic force microscopy dynamic force spectroscopy optical tweezers nanofabrication methods self-organization fluorescence microscopy optical spectroscopy

University of LinzAustria

Peter HinterdorferPurdue 10-05-2006 50

single molecule recognition atomic force microscopy

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