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

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

Artificial GroEL/ES Cage

Spherical cage connected via periodic water channels

Nonpolar : CH2 Polar : NH, CO Repulsive : CY

Combination of 3 surface types6 nm

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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.

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Example 1: Cage refinement of Rosetta model of 1SRO

Start Finish

Refolding in repulsive cageFan Hao

Example 1: Cage refinement of Rosetta model of 1Sro

Experimental Model

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Cage refinement of Rosetta model of 1SRO

Initial Refined X-ray

RMSD2nd structure

RMSD2nd structure

Example 1: Cage refinement of Rosetta model of 1Sro

Start Finish

100 ns

Refolding in repulsive cageFan Hao

Example 2: Refinement of 1VCC

X-ray structure ROSETTA model

RMSD 0.60 nm

N-terminus

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helix behindhelix in front

RMSD2nd structure

Initial Refined X-ray

Example 2: Cage refinement of 1VCC

RMSD2nd structure

correct -sheet

helix lost

Example 3: Cage refinement of Rosetta alternative model of 1AFI

Experiment Model

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Start Finish

Refolding in repulsive cageFan Hao

Example 3: Cage refinement of Rosetta model of 1afi

• 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

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.

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