Pinze ottiche

Ciro CecconiDipartimento di Fisica, Informatica e Matematica

Università degli Studi di Modena and Reggio Emilia

14 dicembre, 2017

UNIVERSITA’ DEGLI STUDI

di Modena e Reggio Emilia

E-mail: ciro.cecconi@unimore.it

Sommario

� Cenni storici sulle pinze ottiche

� Fisica dell’intrappolamento ottico

� Setup pinze ottiche

� Procedure sperimentali per la mipolazione di

molecole singole con pinze ottiche

� Studio del protein folding con pinze ottiche

History of optical tweezers

Most of the early work in this field was done by Arthur Ashkin

of Bell Labs.

1986 A. Ashkin, J.M. Dziedzic, J.E. Bjorkholm, and S. Chu

“Observation of a single-beam gradient force optical trap

for dielectric particles“, Optics Lett., 1986. First observation

of 3D all-optical trap (birth of optical tweezer). A single laser

focused through a microscope was used to trap polystyrene

balls with diameters 10 µm to 25 nm [2].

1987 Ashkin, A. and J. M. Dziedzic. “Optical Trapping and

Manipulation Of Viruses and Bacteria” Science, 1987.

1987 Ashkin, A., J. M. Dziedzic and T. Yamane. “Optical Trapping

and Manipulation Of Single Cells Using Infrared-Laser Beams.”

Nature 1987.

Early 1990s and afterwards, researchers like Steven Block,

James Spudich, Carlos Bustamante, ..

Varie applicazioni in fisica e chimica, ma soprattutto in biologia.

Applicazioni delle pinze ottiche

1) Proprietà meccaniche delle cellule

2) proprietà meccaniche di biopolimeri quali la cromatina e DNA

3) Processi di ripiegamento e denaturazione di proteine e di molecole di RNA

4) studio del meccanismo di funzionamento dei motori molecolari

(DNA polimerasi, RNA polimersai, elicasi etc.)

In 1619 Johannes Kepler suggested that the solar repulsion of the finely divided matter of comet tails was due to the pressure of light

Light exerts force on matter

Intrappolamento ottico

Laserbeam

Pin

Pout

∆P1net ∆P

F = -dP/dt

Reflected rays

∆P2

gradient force

F= dP/dt

∆P

Laserbeam

Pin

Pout

∆P1

net ∆P

F = -dP/dt

∆P2

gradient force

Laserbeam

F=-dP/dt scattering force

(Pfotone = h/λ)

lens

forcereflectedrays

force

∆P

∆P

F

F

Single-beam trap

reflectedrays

Intrappolamento ottico

Dual-beam-trapDual-beam trap

Intrappolamento ottico

Detecting external force from changes in light momentum

∆X

External

force

Laser

beam

Optic

axis

photo

detector

Smith et a. Science (1996)

F=(W/c)( ∆x/RL),

W = l’intensità della luce,

c = la velocita’ della luce,

∆x offset misurato dal fotorivelatore di posizione

RL = la lunghezza focale della lente

OB

JO

BJ

LA

SE

RL

AS

ER

pipette

DNA

position detector

liquid chamber

position detector

qwppbs

Double-Beam Force Measuring Laser Tweezers

Laser tweezers

Force-extension curves

Optical tweezers set up with different geometries

Main optical tweezers components

Laser

Laser

Optical fiber

objective

Photodetectors

Fluid chamber

Piezoelectric actuator

Optical tweezers setup

Manipolazione meccanica di biomolecole

Laser

pipette

Globular proteinDNA handles

biotinstreptavidin

digoxigeninantibodies

SH

SH

SH

SH

DNA

Thiol

chemistry

Molecule tethering

long molecule

(λ DNA, titin)

-SH

-SH

155

4

Folding core

RNaseH

Q4C/V155C*RNase H

Synthesis of a cysteine-bearing proteinand DNA handles

PCR synthesis

biotin

digoxigenin

2,2'-Dithiodipyridine

disulfideCysteine-modified

protein

+2

2

Thiol-pyridine-activated

protein

Pyridine-2-thione

SH

+

2

SS

1)

2)

SHHS

S N

SSN

NS

SN S S N

SSN S S N S N

S S

SHDNA

DNA-protein coupling

0.1

0.2

0.3

0.4

0.5

0

Ab

s (

34

3 n

m)

2 4 6 8 10 14120

Time (min)

0.6

0.5

0.4

0.3

0.2

0.1

0.0

0 10 20 30 40 50 60

2,2'-Dithiodipyridine

disulfideCysteine-modified

protein

+2

2

Thiol-pyridine-activated

protein

Pyridine-2-thione

1) SHHS

S N

SSN

NS

SN S S N

Following protein derivatization photometrically

0.01

0.07

0.06

0.05

0.04

0.03

0.02

Abs

(343 n

m)

0 200 400 600 800 1,000 1,200

Time (min)

SH

+

2

SS

2)S

SN S S N S N

S S

SHDNA

Following protein-DNA coupling photometrically

single handles

-S-S-

-SH-S-S-

-S-S- -S-S-

protein

Gel electrophoresis analysis of protein-DNA complexes

molecular markers

Atomic force microscopy of DNA-protein complexes

0.7 um

0.7 um 0.85 um

0.7 um

Experimental set-up

piezoelectric

actuator

10-15 min

Experimental strategy

digbio

proteina

piezoelectric

actuator

Bead trapping

Muscles

Extracellular matrix

Cytoskeleton

Many proteins in nature are subject to mechanical force

mechanosensors

Proteosome

Protein folding is an heterogeneous process

Raschke et al. Nature Structural Biology (1997)

Ribonuclease H (RNase H)

An intermediate is formedwithin 12 msec

CD

sig

na

l

Time (sec)

0.0

-2.5

-5.0

-7.5

-10.0

0 5 10 15

unfolded signal

folded signal

∆G(UI)=3.6 ±0.1 kcal/mol

Bi-phasic folding

Enzyme that degrades RNA when RNA is hybridized with ss DNA

It is made of 155 amino acids

Unanswered questions

U <=> I --> N

N

‡

UI

N

‡

UUII

On-pathway

I <=> U --> N

N

‡

UI

N

‡

UUII

Off-pathway

Is the transition from U to I

a two state process, or a multistate process?

U I U II3I2I1

I

U N

N

‡

UI

N

‡

UI

Parallel pathway

Is I obligatory?

Is I on- or off-pathway?

-SH

-SH

155

4

Folding core

RNaseH

Q4C/V155C*RNase H

Synthesis of a cysteine-bearing proteinand DNA handles

PCR synthesis

biotin

digoxigenin

Experimental set-up

Reversible work

equal to the change

in free energy of the

system

Extension (nm)0

5

10

15

20

25

30F

orc

e (

pN

)

50 nm

N U

UI

Force-extension curves

Native

structureRandom

coiled polymerExtended structure

∆Gbulk ∆Gstretching

Correcting for the stretching of the unfolded state

+−

−=

−

cc

B

L

x

L

x

p

TF

4

11

4

12

κ

Bustamante et al. Nature, (1994)

Worm-like chain model

W=∆Gstretching=5.7 Kcal/mol

Reversible work

equal to the change

in free energy of the

system

Extension (nm)0

5

10

15

20

25

30F

orc

e (

pN

)

50 nm

N U

UI

Free energy change of the U I transition

∆G(UI) = 4 ± 2.6 Kcal/mol

After correcting for the stretching of the unfolded state

Fluctuations between U and I

“Hopping” between U and I

6.1 pN

6.0 pN

5.58 pN

5.4 pN

4.8 pN

1 sec20 nm

U

I

The inverse of

the life - times

in the I and U

states give us

the rate coeff.

for the reaction

as a function of

force

( ) ( )

Tk

GG

Tk

xFFK

B

strUI

B

UI

eq

∆+∆−

∆=

0

)(ln

( ) ( )0ln0

eqBUI KTkG −=∆

( )

( ) nm68.125.11x

mol/Kcal24G

Values Fit Best

UI

0

UI

±=

±=

∆

∆

Bustamante et al.Ann. Rev. Biochem(2004)

Measuring Keq of the U I transition

Bulk experiments Laser Tweezer

experiments

∆G0(UI)

3.6 ± 0.1 kcal/mol 4 ± 2.6 Kcal/mol

4 ± 2 Kcal/mol

Comparison between bulk and in single molecule studies

Mechanical unfolding of RNase H (I53D)

WT

50 nm0

5

10

15

20

25F

orc

e (

pN

)

Extension

6.1 pN

6.0 pN

5.58 pN

5.4 pN

4.8 pN

The U I transitionappears to be a twostate process

Is the U I transition a two state process?

U

I

Is I an on- or off-pathway intermediate?

654 656 658

Exte

nsio

n (

nm

)

2890

2900

2910

Time (sec)

N

TS2

ITS1

U

G

Reaction Coordinate

~4.5nm

Fo

rce

Extension

I is an on-pathway intermediate

N

‡

UI

N

‡

UUII

Is I an obligatory intermediate?

I

U N

N

‡

UI

N

‡

UI

Parallel pathway

Extension (nm)0

5

10

15

20

25

30

Forc

e (

pN

)

50 nm

I is an obligatory intermediate

Cecconi et al. Science (2005)