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Assisted Folding: Chaperone Cage
1. Encapsulation(prevent aggregation)
2. Preferential binding of unfolded states (drives local unfolding; exposed hydrophobic surfaces)
3. Release and refolding in a sequestered environment.(confinement limits conformational space; compact native states)
4. Multiple cycles(energy dependent)
Experimental approaches to promote folding: low protein concentration; varying levels of denaturant; space-filling molecules;
multiple folding cycles.
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GroEL/ES (~8000 a.a.) + 1Sro (76 a.a.)
14 nm
~3 nm ~8 nm ~7 nm
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I
I
ADP ADP I
ATP ATP
I N
ADP ADP ADP ADP
ATP ATP
NI
7 ATP
7 ADP
7 ATP
7 ADP
~ 10 s
ATP Driven Binding and Release
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Artificial GroEL/ES Cage
Spherical cage connected via periodic water channels
Nonpolar : CH2 Polar : NH, CO Repulsive : CY
Combination of 3 surface types6 nm
Fan Hao
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Stage 1: Binding and unfolding
Nonpolar : CH2 Polar : NH, CO
diameter6 nm
Artificial GroEL/ES Cage
NB: Protein placed in centre of cavity.
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Stage 2: Release and refold
a) explicit water
Type 1: Mixed hydrophobic/polar (low density)
Type 2: Repulsive
b) chaperone cage: surface type 1 and 2
Artificial GroEL/ES Cage
NB: Protein placed in centre of cavity.
Fan Hao
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Example 1: Cage refinement of Rosetta model of 1SRO
Start Finish
Refolding in repulsive cageFan Hao
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Example 1: Cage refinement of Rosetta model of 1Sro
Experimental Model
Fan Hao
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Cage refinement of Rosetta model of 1SRO
Initial Refined X-ray
RMSD2nd structure
RMSD2nd structure
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Example 1: Cage refinement of Rosetta model of 1Sro
Start Finish
100 ns
Refolding in repulsive cageFan Hao
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Example 2: Refinement of 1VCC
X-ray structure ROSETTA model
RMSD 0.60 nm
N-terminus
Fan Hao
helix behindhelix in front
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RMSD2nd structure
Initial Refined X-ray
Example 2: Cage refinement of 1VCC
RMSD2nd structure
correct -sheet
helix lost
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Example 3: Cage refinement of Rosetta alternative model of 1AFI
Experiment Model
Fan Hao
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Start Finish
Refolding in repulsive cageFan Hao
Example 3: Cage refinement of Rosetta model of 1afi
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• Mixed hydrophobic/hydrophilic surfaces most effective in driving unfolding.
• Confinement (repulsive surface) improves efficiency of refolding.
• Surfaces show differential interaction with secondary structurei.e. helices destabilized, β-sheet formation favoured.
• Insufficient discrimination between native and none native structure.
Assisted Folding: Chaperone Cage
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Can we refine all structures? No
• Accuracy of the force field.
• Need for statistically relevant samples.
• Need for alternate approaches to drive refolding.
• Not all information in structural databases is equally reliable.
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PDBID Exp. structure5ns MD
side-chains rebuilt 5ns MD
1aoy 0.45 0.35
1stu 0.29 0.30
1vif 0.29 0.29
1sro 0.30 0.29
1tuc 0.24 0.18
1sap 0.36 0.31
1afi 0.17 0.18
1bb8 0.70 0.73
1vcc 0.26 0.16
2bby 0.26 0.31
2fmr 0.52 0.35
1a1z 0.27 0.36
1ail 0.27 0.35
1bw6 0.71 0.50
1cei 1.14 0.57
1coo 0.23 0.67
1lea 0.23 0.26
1rpo 0.80 0.84
2af8 0.67 0.60
2ezh 0.30 0.29
Root mean square positional deviation from the experimental structure
after 5 ns simulation.
1. Original NMR or X-ray2. After rebuilding side-chains
After deleting and rebuilding side-chains 4 get worse > 0.05 nm7 get better > 0.05 nm
Dimer
Dimer