Exploring photolytic triggers of protein folding

1
Exploring photolytic triggers of protein folding
Jon Waltho Department of Molecular Biology
Biotechnology The University of
Sheffield Future Prospects for
Macromolecular Dynamics on 4GLS Daresbury, 26-27
January 2007
2
Protein Denaturation and Refolding
High Denaturant
Folded State
Unfolded State
U
Measured Parameter
F
F
U
Low Denaturant
m-values correlate strongly with the degree of
burial from solvent of hydrophobic residues
Denaturant concentration
Folding
Unfolding
Slope "m value"
Free Energy
F
U
Denaturant concentration
3
Two-state Folding Funnels
Smooth
Golf-course
Bumpy bowl
(Dill Chan 1997, Nature Struct. Biol. 4, 10-19)
4
Observation of Kinetic Intermediate States
High Denaturant
U
I
F
F
I
Refolding
U
Low Denaturant
Unfolding
m-values correlate strongly with the degree of
burial from solvent of hydrophobic residues
Slope "m value"
Free Energy
F
I
U
Denaturant concentration
5
Free Energies of Folded and Kinetic Intermediate
States relative to Unfolded States
6
Three-state Folding Funnels
Stabilisation by a wide range of native contacts
Stabilisation by a narrow range of native
contacts
Somewhere in-between
(Dill Chan 1997, Nature Struct. Biol. 4, 10-19)
7
Photocleaving Restrained Non-Native States
Crosslink incompatible with native fold
Photocleavage
Monitor refolding
8
Experimental Folding Models
- Phosphoglycerate kinase (PGK) - CD2 d1 domain
- Protein G
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bsPGK (W290Y) Domain Folding
W315

• Each domain forms an I-state
• independently
• N-domain folds first
• Slowest step is C-domain
• I-F transition

(Parker et al., Biochemistry (1996) 35,
15740-15752)
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Probing of Rare States using Amide Exchange
kopen
kint
CH
OH
OD
kclose

• Kinetic Intermediates
• CD2 Parker et al., Nature
• Struct. Biol. (1998) 5194

Closed Form
Open Form
?G
kopen
kclose
kobs
. kint
kopen
kclose
kint
F
O

• Partially Folded States
• PrPc Hosszu et al., Nature
• Struct. Biol. (1999) 6740

C
?m

• Molten Globules
• bPrP Jackson et al., Science (1999) 2831935

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Protection Factors in I state of intact PGK
2H,13C,15N-labelled PGK pH 7.5, 310K
(Hosszu et al., Nature Struct. Biol. (1997) 4,
801-804)
1 174
371 394
(Reed et al., J. Mol. Biol. (2003) 330, 1189-1201)
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Photocleavable crosslinked versions of N-PGK
Mutations F17W, C18V E108C, R118C
Choice of Attachment Site for Crosslinker -
high effective molarity of cys residues -
sufficient degree of fold disruption - cleaved
residue tolerated in folded state
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Protocol for Crosslinker Attachment
Step 1 modification of both Cys
Step 2 unfolding and exposure of the crosslinker
Step 3 oxidation and refolding
verification of product
Electrospray MS
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Testing the Authenticity of Crosslinkage
Electrospray MS
Crosslinked protein
Cleavage of crosslink via disulphide reduction
Verification of crosslink reduction via
derivatisation ( 250 Da 2 x maleimide)
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Characterisation of Crosslinked C108,C118-N-PGK
Circular Dichroism
Trp Fluorescence
Reduced
Crosslinked
Crosslinked
Reduced
Crosslinked
Crosslinked
220 nm
330 nm
Reduced
Reduced
GuHCl
GuHCl
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1H NMR of Crosslinked C108,C118-N-PGK
Top Crosslinked Middle Reduced
Crosslinked Bottom Prior to Crosslinking
1.0 M GuHCl
Crosslinked E108C,R118C-N-PGK is a molten
globule in the absence of denaturant
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Identification of molten globule states
Folded Protein
ppm

• Molten Globule
• reduced resonance dispersion
• resembles unfolded state

ppm
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Chemical shift changes in the denatured state
1H,15N HSQC crosspeaks versus GuHCl concentration
Red 1.3 M Black 1.5 - 1.9M Blue 2.0 - 4.0 M
x-axis 0.2 ppm 1H y-axis 2.0 ppm 15N
Resonance assignment 20 kDa, 174 residues, pH
6.0, 25oC, 0.8 mM protein, 4M GuHCl
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Comparison with SEC, CD and FL data
N-PGK

• High denaturant transition does not involve large
  scale collapse of the unfolded state
• Rate gt 104s-1 mU-I1 lt 2 M-1
• Lower denaturant transition does involve
  collapse
• Rate 103-104 s-1 mU-I2 lt 5 M-1

GdnHCl
(mU-Ikin 11 M-1 mU-F 16 M-1)
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Conformations in the denatured state
- Denatured state helices are longer than those
in the native state
- RDCs indicate parallel or antiparallel
- 4/5 helices in low denaturant concentration,
denatured state
Ikin
F
U
I2
(Reed et al., J. Mol. Biol. (2006) 357, 365-372)
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Experimental Folding Models
- Phosphoglycerate kinase (PGK) - CD2 d1 domain
- Protein G
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CD2 Domain I Folding behaviour
Kinetics of folding/unfolding at pH 5, 7 and 9
kF-I by NMR
KF/I 3.4x104 KI/U 8.0x101
Ig-V fold
kI-F 6.7 s-1 kF-I 2.0x10-4 s-1
98 residues No disulphides
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CD2 Competition vs Equilibrium Amide Exchange
U
2
Competition
log10PI
0
I
U
I
4
Equilibrium
log10PF
2
0
F
20
40
60
80
-Values extrapolated to EX2 limit -Filled Bars
EX1 limit at pH 9.0
(Parker et al., Nature Struct. Biol. (1998)
5194-198)
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CD2 Domain I Comparison of equilibrium and
competition methods
White Exchange controlled by kF-I in
equilibrium experiment
B
E
F
E
B
C
F
C
C
E
F
Red Exchange protected in competition
experiment
B
Most Ig folds have a disulphide bond between
strands B and F
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CD2 Domain I Double Cysteine Mutants
13 Double thiol mutations distributed throughout
the fold
G13C,K66C
E33C,L38C
N15C,K64C
T75C,D94C
L58C,D62C
R31C,E41C
T79C,N90C
E29C,K43C
Y81C,R87C
M23C,N60C
Also Core Mutants I18C,V78C A40C,L50C
I57C,L63C
(Mason et al., Biochemistry (2002) 4112093-12099)
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Crosslinkage of C29,C43-CD2.d1
E29C,K43C
E29C,K43C
C29,C43 disulphide links strand C and C
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Characterisation of Crosslinked C29,C43-CD2.d1
Circular Dichroism Denaturant Dependence
Crosslinked
Non-crosslinked
GuHCl M
Wavelength (nm)
- Crosslinked C29,C43-CD2.d1 is a beta-rich
molten globule - A beta-rich molten globule
component is apparent in the denatured state of
the non-crosslinked protein
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Experimental Folding Models
- Phosphoglycerate kinase (PGK) - CD2 d1 domain
- Protein G
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Protein G Double Cysteine Mutants
2-State Folding Behaviour
T25C,N37C
T25C,D46C
Current Double Cysteine Mutants - C25,C37
Distortion of Helix - C25,C46 Separation of
Helix and Sheet
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Testing the Authenticity of Crosslinkage
MALDI MS
7736
7738
(Crosslinked C25,C46-Protein G NEM)
Crosslinked C25,C46-Protein G
7739
Cleavage of crosslink via disulphide reduction
7988
Verification of crosslink reduction via
derivatisation ( 250 Da 2 x NEM)
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1H,15N-HSQC NMR shows residual structure in
crosslinked C25,C46-Protein G
1H,15N-HSQC in water (Mixture of crosslinked and
non-crosslinked species)
15N (ppm)
Blue peaks Crosslinked Red peaks Non
crosslinked
1H (ppm)
- Crosslinked C25,C46-Protein G is a molten
globule in water - Crosslinked molten globule
unfolds before reduced folded form
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Photolysis Choice of Restraining Agent
Aminothiotyrosine
Amino aryl disulphide derivatives based on the
work of Volk, Hochstrasser and DeGrado
(Lu et al., J. Am. Chem. Soc. 1997, 119,
7173-7180 Volk et al., J. Phys. Chem. B 1997,
101, 8607-8616)
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Photocleavage of model crosslinkers

DOD
time (ps)
Cleavage 100 fs pulse (260 nm 100
nJ) Detection Absorbance (520 nm)
(Milanesi et al. 2004, Chemistry 10, 1075-1710)
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Photocleavage of C108,C118-N-PGK crosslinked
with p-maleimidothiophenyl groups
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Summary
The next phase for photocleavage - Higher
sensitivity detection of folding transitions -
Lower recombination of cleaved products
Photocleavable crosslinks can be readily
introduced PGK Large a/b fold with
stable Ikin state and equilibrium MG state
CD2 Medium sized all b fold with
metastable Ikin state Protein G Small
a/b protein that folds without a stable Ikin
state
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Acknowledgements
University of Sheffield Lilia Milanesi
Dominic Sloane Clare Jelinska Chris
Hunter Jeremy Craven Rosie Staniforth
Andrea Hounslow Tooba Alizebah Laszlo Hosszu
Martin Parker Matthew Cliff Michelle Reed
Andrew Splevins Karl Syson
University of Bristol Tony Clarke University
of Leeds Godfrey Beddard Gavin Reid Daniel
Shaw University of Liverpool Martin Volk
Sagarika Dev Daresbury Laboratory Dave Clarke
Gareth Jones