Disturbing (polymers and) proteins with Atomic Force Microscopy

José L. Toca-Herrera

ETSEQ, 02/06/2004

DNA, proteins are polymers...

with AFM you can see them (nano)structure!

and most important you can measure inter and intramolecular forces

Is that all you can do??

The AFM Experiment: imaging

The movements of the reflected light are "seen" by a photodiode. So the magnitude of the diode current in addition to the position of the tip on the plane leads to an image, which provides three-dimensional information regarding the sample

The AFM Experiment: pulling

Cantilevers (and tips)

Different types and shapes different mechanical properties

The cantilever is the sensor of the AFM

Mechanical properties can tell us something about interfacial phenomena

What does AFM have to offer?

• Alternative to conventional methods of denaturation (e.g. heat, acid, chemical denaturant) and kinetic reactions in general

• Single molecule experiment: measurement of inter and intra molecular forces

• Well defined reaction coordinate• Direct comparison with all-atom MD and

Monte Carlo simulations

Something more to offer?

• Alternative to conventional methods of “take a chance on me”: other microscopies like TEM, SEM…sub-nanometer resolution

• You can follow processes on-line such as structural changes

• NOT only single molecule: attach a colloidal particle

• You can work with(in) solvents: biological condition, etc…

S-layer on PAA

- Silicon wafer + PEI + (PAA + PDADMAC)2 + PAA

60nm60nm

Switching off/on S-layers : water-EtOH mixtures

- Hydrophilic Silicon as substrate

- 80% vol EtOH unfolds the protein...after buffer treatment we get it back!

Modeling: Monte-Carlo Simulations (I)

• Folding and Unfolding rates given by

0)exp()0(

))(exp()( *

ff

fDTSf

Fxk

FxGFk

)exp()0(

))(exp()( *

uu

uNTSu

Fxk

FxGFk

In the Beginning there was Titin…

Interpreting traces

• Unfolding proteins by AFM is a kinetic measurement: average unfolding force depends on pulling speed.

• Average unfolding rates can be estimated by Monte-Carlo simulation.

Conservative, non-disruptive, significantly destabilising

A’-strandV13A

G-strand

V86A

F strandF73L

C-D loop

L41A

Choice of mutants is critical

A-strand

V4A

Evidence from mutation

When we pull a protein with a destabilising mutation in the A strand (V4A) it does not affect the unfolding forces at all

MD simulation supports this pattern

Carbohydrate-protein interaction

-4

-2

0

2Forc

e (n

N)

-300-200-1000 Extension (nm)

120 nm

300 pN

95 nm

0 500 1000 1500 2000 2500 3000 3500 40000

10

20

30

40

50

Fre

quen

cy

Force

going away from the surface

a) Attracttive forces appear when the tip approaches the surface covered with S-layers.

b) By cantilever retraction large adhesion forces with different peaks can be measured (see histogram, left). After pulling a length of about 120-140 nm, here we are measuring carbohydraete/protein forces... elasticity of protein domains can also be detected (in general lower than 300 pN).

Force between cell walls

-SiOx coated particle glued (UHU-plus endfest 300) on rectangular cantilever-Adhesion force about 1mN/m-Conexion with SFA

1.0 1.5 2.0 2.5 3.0 3.50

2

4

6

8

10

Freq

uenc

y

Force (nN)

Conclusions

AFM is a suitable tool to investigate polymer elasticity, binding kinetics, molecular recognition and surface properties…

and

…this includes everything related with biology, surface chemistry, physics and material science

AFMs are in continuos development: hardware and software…almost everybody can contribute!