dipartimento di fisica, informatica e matematica università degli...
TRANSCRIPT
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: [email protected]
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)