Jane Clarke May 2004
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Transcript of Jane Clarke May 2004
Jane Clarke
May 2004
How do proteins withstand force?
Examining the effect of force on a protein
folding landscape by combining atomic
force microscopy, protein engineering and
simulation
How do proteins withstand force?
Examining the effect of force on a protein
folding landscape by combining atomic
force microscopy, protein engineering and
simulation
Robert Best
Susan Fowler
Annette Steward
Kathryn Scott
José Toca Herrera
Cambridge University Dept of ChemistryMRC Centre for Protein Engineering
Cambridge University Dept of ChemistryMRC Centre for Protein Engineering
Wellcome Trust & MRCWellcome Trust & MRC
Emanuele Paci (Zurich)Martin Karplus (Strasbourg/ Harvard)
Phil Williams (U. Nottingham)
Gene
The Protein Folding ProblemThe Protein Folding Problem
Misfolded protein
sequence A
sequence B
Folded protein
Unfolded
protein
Proteins fold co-operatively into a unique 3-dimensional structure that is the most stable conformation
Proteins fold co-operatively into a unique 3-dimensional structure that is the most stable conformation
functionWhy? How?Why? How?
Why not?Why not?
How?How?
But proteins don’t just fold one time and that’s it.
Mechanical unfolding of proteins may be important in translocation and degradation
and in mechanically active proteins
For some proteins resisting unfolding may be important
But proteins don’t just fold one time and that’s it.
Mechanical unfolding of proteins may be important in translocation and degradation
and in mechanically active proteins
For some proteins resisting unfolding may be important
Protein folding pathways - and landscapesProtein folding pathways - and landscapes
How does force modify the unfolding landscape? How does force modify the unfolding landscape?
Karplus, Dobson
D
I
N
‡2
‡1
We can explore the unfolding landscape
by folding and unfolding experiments
N
TS
D
∆GTS-N
xu
xf
∆∆GTS-N
∆∆GD-N
When you add FORCE (F):Relative to the native state, N,the barrier to unfolding (∆GTS-
N) is lowered by: Fxu
and the free energy of unfolding (∆GD-N) is lowered by:
F(xu + xf)
The protein is less stable and unfolds more rapidly - the unfolding rate (ku) is a measure of the height of the barrier between N and TS
What does AFM offer?What does AFM offer?
• Can investigate the way the energy landscape is perturbed by force
• Known reaction co-ordinate (N-C length) making it easier to do direct comparison with simulation
• Single molecule experiments offer the possibility to observe rare events
The AFM ExperimentThe AFM Experiment
Asylum Research MFP
1
2
3 4
∆L
F
1. Non-specific adhesion 2. Unfolding of one domain
3. Unfolded protein stretching 4. Protein detaches
• Unfolding proteins by AFM is a kinetic measurement: mean unfolding force depends on pulling speed.
• Unfolding rate constant (extrapolated to 0 force) (ku
0) and unfolding distance (xu) can be estimated by Monte Carlo simulation or analytical techniques.
Analysis of AFM dataAnalysis of AFM data
Fo
rce
(N
)
Gaub, Fernandez, Evans
Slope givesxu
Intercept givesku
0
Interpreting the traces:Which traces to choose?Interpreting the traces:
Which traces to choose?
The basic reminders about kinetics and thermodynamics
The basic reminders about kinetics and thermodynamics
Force measurements of protein unfolding are kinetic measurements not thermodynamic measurements
So… Beware of the word “stability” - what does it mean in the context of force measurements?
“In a protein made up of a number of domains the least stable domains will unfold first and the most stable domains will unfold last”
Titin I27 is significantly more stable than I28 (7.6 vs. 3.2 kcal/mol) but I28 unfolds at significantly higher forces
But…It is not possible to determine the stability of a protein using AFM
folding and unfolding data
The basic reminders about kinetics and thermodynamics (2)
The basic reminders about kinetics and thermodynamics (2)
€
ΔG = −RT lnkukf
⎛
⎝ ⎜
⎞
⎠ ⎟
Carrion Vasquez
It is possible to measure refolding rates using AFM
BUT - the unfolding and refolding pathway are not necessarily (are unlikely to be?) the reverse of each other.
Titin - an elastic proteinTitin - an elastic protein
M-LINENNNNZ-LINEZ-LINEPEVKIgFnIIIM-BANDI-BANDI-BAND1 µm
Titin - effect of forceTitin - effect of force
Very low force Protein domains straighten out
“working” forces Unstructured region unfolds
Very high force One or two domains unfold toprevent the protein breaking
First experiments - using whole proteins with heterogeneous domains
First experiments - using whole proteins with heterogeneous domains
Gaub, Bustamante, Symmonds, Fernandez
How do titin domains resist force?How do titin domains resist force?
Can we characterise the titin I27 forced unfolding pathway in detail?
Can we characterise the titin I27 forced unfolding pathway in detail?
To make multiple repeats of one titin domain
A tag to allow easy purification
Using molecular biologyUsing molecular biology
A tag to allow attachment to
AFM
In simulations the first step is to form an intermediate by detachment of the A-strand
In simulations the first step is to form an intermediate by detachment of the A-strand
Fernandez, Schulten
Humps?
V4A
When we pull a protein with a destabilising mutation in the
A-strand (V4A) it does not affect the unfolding forces at all
Fowler et al. JMB 2002 322, 841
This intermediate is stable and has essentially the same structure as the
native state
This intermediate is stable and has essentially the same structure as the
native state
1 H
15N
Increasing force
0 pN >100 pN≈100 pN
N
N
N
I
II
‡F
‡F
‡F
3 Å
ku≈10-4
∆G ≈ 3 kcal mol-1
ku is the unfolding rate of I to ‡and xu is the distance between I & ‡
I is populated above 100pN
Titin forced unfolding pathwayTitin forced unfolding pathway
Native stateN
IntermediateI
Transition State
?
UnfoldedD
N
I
‡
Free energy profile under force
A’
GG
AA’
N
C
G GG
N NC
C
~3 Å
ku
Using protein engineering to analyse forced
unfolding pathways:
A mechanical -value analysis
Using protein engineering to analyse forced
unfolding pathways:
A mechanical -value analysis
Best et al. PNAS 2002 99, 12143
V4
V13
V86
I23 F73
L41L60
L58
N
C
A
A´
G
F
B
E
D
C
G
Theory
Protein engineering analysis - = 1
Theory
Protein engineering analysis - = 1
N
I
‡
•The unfolding force reflects the difference in free energy between I and ‡
U • If the mutation removes a side chain that is fully folded in the
transition state it will not affect the unfolding force at all.
Protein engineering analysis - = 0Protein engineering analysis - = 0
N
I
‡
•If the mutation removes a side chain that is fully unfolded in the transition state it will reduce the unfolding force by a significant
amount - that we can predict
U
•The unfolding force reflects the difference in free energy between I and ‡
NB only works if the barrier we are examining is the same in WT & mutant(xu must remain the same)
NB only works if the barrier we are examining is the same in WT & mutant(xu must remain the same)
0
50
100
150
200
250
1 10 100 1000 104
V13A
Force (pN)
Pulling Speed nm-s
= 0
WT( = 1)
The A’ strand is partly detached in ‡The A’ strand is partly detached in ‡
0
50
100
150
200
250
10 100 1000 104
F73L
Force (pN)
Pulling Speed nm-s
Most -values are ≈ 1Most of the protein is intact in the transition state
Most -values are ≈ 1Most of the protein is intact in the transition state
0
50
100
150
200
250
1 10 100 1000 104
L41A
Force (pN)
Pulling Speed nm-s
0 50 100 150 200 250
Frequency
Unfolding force (pN)
-90 0
-80 0
-70 0
-60 0
-50 0
pN
7 00 6 50 6 00 5 50n m
70 0
60 0
50 0
40 0
30 0
pN
40 0 3 50 30 0 2 50n m
A mutation in the G strand causes the protein to unfold at lower force
V86A 300 nm/swild type 300 nm/swildtypeV86A
BUTxu changes - can’t do a -value analysis
but this mutation clearly lowers the force, ie must be <1
BUTxu changes - can’t do a -value analysis
but this mutation clearly lowers the force, ie must be <1
0
50
100
150
200
250
10 100 1000 104
V86A
Force (pN)
Pulling speed (nm/s)
Results:The only part of the protein completely detached
in ‡ is A strandA’ and G are partly disrupted. How?
Can molecular dynamics simulations help?
Results:The only part of the protein completely detached
in ‡ is A strandA’ and G are partly disrupted. How?
Can molecular dynamics simulations help?
MD simulations - the protein unfolds via an intermediateMD simulations - the protein unfolds via an intermediate
QuickTime™ and aYUV420 codec decompressorare needed to see this picture.
Analysing structures from the simulations - experimental -values are reproduced
Analysing structures from the simulations - experimental -values are reproduced
00.20.40.60.8
1
00.20.40.60.8
1
00.20.40.60.8
1
45 50 55 60 65rNC
(Å)
00.20.40.60.8
1
4
13
82
86
constant pulling force=350 pN
Proportionof native contacts
intermediate transition state
00.20.40.60.8
1
00.20.40.60.8
1
00.20.40.60.8
1
45 50 55 60 65r
NC (Å)
00.20.40.60.8
1
4
13
82
86
constant pulling force=350 pN
V13
V4
Native State
Transition State
Step 1:A-strand
pulled off to form I
Step 2:G-strand pulled
off, breaking main chain &
sidechain contacts with A’
& sidechain contacts with A-B loop. A’ loses contacts with G
& E-F loop
C
N
N
C
G- strand
A’A
Mechanical unfolding pathway:Mechanical unfolding pathway:
N I ‡ D
Transition State
‡
Native stateN
IntermediateI
UnfoldedD
A’
GG
AA’
N
C
G GG
N NC
C
Titin forced unfolding pathwayTitin forced unfolding pathway
N
I
‡
Free energy profile under force
Increasing force
0 pN >100 pN≈100 pN
N
N
N
I
II
‡F
3 Å
‡F
‡F
ku≈10-4
∆G ≈ 3 kcal mol-1
Force induced unfolding pathway
Increasing force
0 pN >100 pN≈100 pN
N
N
N
I
II
‡F
N
‡D‡F
‡F
ku≈10-4
ku≈10-4
Denaturant (0 pN)
Does force change the energy landscape?Does force change the energy landscape?
Do these transition states have the same structure?
3 Å
Force
The A’ G region remains partly
folded in TS
The A strand unfolds very
early
The core plays no role in
withstanding force and
remains fully folded in TS
The core is partly unfolded
in TS
The A’ G region is fully unfolded
in TS
Chemical denaturant
The A strand remains partly
folded in TS
Increasing force
0 pN >100 pN≈100 pN
N
N
N
I
II
‡F
N
‡D‡F
‡F
ku≈10-4
Denaturant (0 pN)
Force changes the energy landscapeForce changes the energy landscape
Transition states have different structures
3 Å?
?
when?
ku≈10-4
0
50
100
150
200
250
300
10-6 10-4 10-2 100 102 104 106
Force (pN)
Pulling speed (nm/s)
0
50
100
150
200
250
300
10-6 10-4 10-2 100 102 104 106
Force (pN)
Pulling speed (nm/s)
0
50
100
150
200
250
300
10-6 10-4 10-2 100 102 104 106
Force (pN)
Pulling speed (nm/s)
Cannot measure at rates below ~100
nm/s
Experimental limitationsExperimental limitations
0
50
100
150
200
250
10 100 1000 104
V86A
Force (pN)
Pulling speed (nm/s)
•These mutants have a significantly longer xu (~6Å) than wt (~3Å)
All these mutants destabilise the protein significantlyAll these mutants destabilise the protein significantly
• BUT These very destabilising mutants have apparently a lower ku than wild type
Simplest model: these mutants are unfolding directly from NSo xu = xN ->‡ & ku = kN-‡
Simplest model: these mutants are unfolding directly from NSo xu = xN ->‡ & ku = kN-‡
Williams et al: Nature 2003
Increasing force
0 pN >100 pN≈100 pN
N
N
N
I
II
‡F
‡F
‡F
3 Å
In wildtypeku is rate constant for unfolding
from I to ‡F
and xu is the distance between I & ‡F
In the mutant V86Aku is rate constant for unfolding from N to ‡F
and xu is the distance between N & ‡F
This will happen if the mutation allows the protein to unfold before I is populated
(by destabilising I &/or lowering the
unfolding barrier ‡F)
6 Å
Increasing force
0 pN >100 pN≈100 pN
N
N
N
I
II
‡F
N
‡D‡F
‡F
ku≈10-4
Denaturant (0 pN)
Force changes the energy landscapeForce changes the energy landscape
Transition states have different structures
6 Å
ku ≈ 10-7
3 ÅWhen?
ku≈10-4
At v. low forces the “physiological” barrier may be the important one
How do titin domains resist force?How do titin domains resist force?
Why are some proteins moremechanically stable than others?
Why are some proteins moremechanically stable than others?
Titin I27(muscle)
Spectrin(cytoskeleton)
T4 LysozymeBarnase
Tenascin fnIII(intracellular matrix)
(enzymes)