Force-spectroscopy
of single proteins
Force as a regulator of:
-protein folding
-enzyme catalysis
Proteins are exposed to mechanical forces in-vivo
extracellular matrix
fibronectin
connector
complex
synapse
P0
Schwann cell
Cytoskeleton:
actin, etc.
tenascin
cell
S-S bonds
are common
fascilin
NCAM
cell-matrix
Basal Lamina
tenascin
fibronectin
etc.
Anchor proteins
Cytoskeleton
Dystrophin
titin
F
ion channels
Cadherin
muscle
We engineer polyproteins with 8-12 repeats
extension (nm)
Pulling a polyprotein at constant force
produces a staircase of elongation events
ubiquitin
time (s)
Tilting the energy landscape of a reaction
k  Ae
0
u
 G0 / kT
W
W*
G0  W
ku  k e
0 W * / kT
u
*
W  Fx
*
x
The probabilistic nature of unfolding
is apparent in the dwell times
A
protein length (20 nm/div)
20 nm
B
 td
four different molecules
pulled at the same force
(110 pN)
F= 100 pN
Time (s)
Each unique folding pathway is still largely hidden
from view due to low time resolution
length (20 nm/div)
At t ~ 1ms we will be able to observe
bond-by-bond details
force (pN)
?
100 ms
too slow
Force dependent protein unfolding
k  k0 e
Fx
k BT
k0= 0.015 s-1
xu= 1.7 Å
MD simulations of I27 unfolding
A`-G patch of I27
F
6 hydrogen bonds
in A`-G patch
F
Unfolding transition state
contains water molecules
Use solvent substitution to probe for
role of water molecules in TS
0 % glycerol
20 % glycerol
30 % glycerol
50 % glycerol
D2O increases G0 but does not change x
H2O
D2O
H2O ΔXU = 0.25 nm
D2O ΔXU = 0.27 nm
Force-dependent chemical
reactions
F
The (I27G32C-A75C)8 polyprotein
F
“Trapped”
residues
“Unsequestered”
residues
F
Mechanical unfolding exposes the
buried disulfide to nucleophilic attack
nucleophile
Identifying single disulfide reduction reactions
I27G32C-A75C
[DTT] = 0 mM
[DTT] = 12.5 mM
Unsequestered unfolding up to disulfide; ~10.5 nm
Single disulfide reduction & trapped unfolding; ~14.2 nm
Force accelerates the rate of disulfide
reduction by DTT
Force dependency measures the elongation of the S-S bond
at the transition state.
54
r= [Trx] r0exp(FΔxr/kBT)
xr=0.34 Å
S-S bond elongation at TS depends on the reducing agent
2.5 mM TCEP
x=0.47±0.03 Å
rate (s-1)
rate (s-1)
12.5 mM GSH
x=0.29±0.06 Å
Force (pN)
Force (pN)
Studying enzyme catalysis is a big challenge
its all done by rearranging atoms
over very short distances....
below 1Å!
distances below 1Å cannot be easily resolved
Probing the chemistry of thioredoxin catalysis
with force
Chemistry:
SN2 attack
of thiolate
anion on
disulfide
Arne Holmgren ; Eur. J. Biochem, 1968, 6:475-484
Arne Holmgren et al; PNAS, 1975, 72:2305–2309
A single molecule thioredoxin system
Identifying disulfide reduction by single Trx enzymes
Trx= 0
Trx= 8mM
The rate of reduction is both force and
[Trx] dependent
[Trx]= 8 mM
F= 100 pN
Trx catalysis has a bimodal force dependency
15:1
k01 = k01(0) [Trx]
k12 = k12(0) exp(FΔx/kBT))
k02 = k02(0) [Trx] exp(FΔx/kBT))
• two pathways for Trx
reduction (I & II)
• Fits reveal a ratio of 15:1
Path I
• Inhibited by force
• complex shortens by
Δx12 = -0.73 Å
k01 = k01(0) [Trx]
k12 = k12(0) exp(FΔx12/kBT))
Nucleophilic attacks
are directional
Reorientation of the
stretched bond is
required to have all
three S atoms in a line
Path II
k02 = k02(0) [Trx] exp(FΔx02/kBT))
• Accelerated by force
• complex elongates by
Δx02 = 0.22 Å
Transition states for SN2 thiolate attack in water.
Pedro Alexandrino Fernandes and Maria Joào Ramos: Chem. Eur. J. 2004, 10, 257 ―266
The P34H mutation reduces ratio I/II
5:1
• reduced k01
unchanged Δx12 and Δx02
Trx mutations can change both a0 and x
1.5
WT 10 µM
-1
rate (s )
2.0
1.0
G74S 10 µM
0.5
P34H/G74S 10 µM
0
200
400
Force (pN)
600
Rate vs Force plots are sensitive to chemical mechanism
well into the sub-Å level. No other technique can do this.
6
hTrx
5
DTT
-1
r (s )
4
3
E. Coli Trx
2
1
0
0
100
200
300
400
Force (pN)
500
600
Dwell times to reduction, in log binned plots
readily uncovers the number of rate constants
Mechanical injury leads to oxidative stress
and high mechanical forces
swelling, inflammation
ROS
F
Oxidative stress
fibronectin
promotes formation
of disulfide bonds, stiffening tissues
Chemical reactions catalyzed by enzymes
are affected by mechanical force, then...
trauma, swelling, inflamation
High forces in vascular damage
Stent placement
Understanding protein folding is one of the
grand challenges of modern biology.
Can we say something new with the AFM?
So far we have mostly studied proteins
using chemical denaturation.
urea
unfolded state (undefined)
folded
fluorescence
(indirect)
reaction coordinate (unknown)
ku  k e
0 m[ D ]
u
time
The force-quench experiment
length (20 nm/div)
3
4
2
1
5
force (pN)
ubiquitin
time (s)
The second pull measures the extent to which
mechanical stability is recovered after folding
The fraction of stable proteins
recovers exponentially
The unstable component elongates in fast steps
The time to recovery is measured from the moment
that the protein has collapsed
Folding is slowed by
force
k0= 100 s-1
xf= 8.2 Å
Identical force quench experiments,
very different outcomes
Folding trajectories
Failures (majority)
After extension, the protein is mostly a very stiff polymer
and those that readily contract typically fold
Every extension resets the dihedral space
to a new starting point
Reduced hydrophobic collapse in a 40 % ethanol solution
Saline
Saline + 40% ethanol
Change in free energy for entropic and
hydrophobic collapse
Entropy
3Å
Hydrophobic
collapse
Energy landscape of an extended protein relaxed to ~10 pN
±180° at Lcontour
Purely entropic
“flat” region
Hydrophobic
forces are strong,
steep funnel
Folding after a force-quench
entropic (chain)
collapse
hydrophobic (solvent)
collapse
folded
unstable
folded
stable
Carmelu Badilla
Kirstin Walther Jasna Brujic
Arun Wiita
Sergi
Garcia
Manyes
Robert Szoszkiewicz
Frauke Grater
Lorna Dougan
Raul Perez-Jimenez
Koti Ainavarapu
A few conclusions
-Force-spectroscopy tracks the conformations
and chemistry of single proteins with sub-Ångstrom
and millisecond resolution (will get better).
-Force probes novel regions of the folding and
unfolding energy landscape of proteins.
-Force regulates the rate of chemical reactions.
And thanks to my collaborators:
-Bruce Berne; Columbia Chemistry
-Jose M. Sanchez-Ruiz, Universidad de Granada
-Arne Holmgren, Karolinska Institute
-Hui Lu; University of Illinois