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)