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 : CH2Polar : NH, CORepulsive : CY Combination of 3 surface types 6 nm Fan Hao
Stage 1: Binding and unfolding Nonpolar : CH2Polar : NH, CO diameter 6 nm Artificial GroEL/ES Cage NB: Protein placed in centre of cavity.
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
Example 1: Cage refinement of Rosetta model of 1SRO StartFinish Refolding in repulsive cage Fan Hao
Example 1: Cage refinement of Rosetta model of 1Sro ExperimentalModel Fan Hao
Cage refinement of Rosetta model of 1SRO Initial Refined X-ray RMSD 2nd structure RMSD 2nd structure
Example 1: Cage refinement of Rosetta model of 1Sro StartFinish 100 ns Refolding in repulsive cage Fan Hao
Example 2: Refinement of 1VCC X-ray structureROSETTA model RMSD 0.60 nm N-terminus Fan Hao helix behind helix in front
RMSD 2nd structure Initial Refined X-ray Example 2: Cage refinement of 1VCC RMSD 2nd structure correct -sheet helix lost
Example 3: Cage refinement of Rosetta alternative model of 1AFI ExperimentModel Fan Hao
Start Finish Refolding in repulsive cage Fan 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 structure i.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.
PDBIDExp. structure 5ns MD side-chains rebuilt 5ns MD 1aoy0.450.35 1stu0.290.30 1vif0.29 1sro0.300.29 1tuc0.240.18 1sap0.360.31 1afi0.170.18 1bb80.700.73 1vcc0.260.16 2bby0.260.31 2fmr0.520.35 1a1z0.270.36 1ail0.270.35 1bw60.710.50 1cei1.140.57 1coo0.230.67 1lea0.230.26 1rpo0.800.84 2af80.670.60 2ezh0.300.29 Root mean square positional deviation from the experimental structure after 5 ns simulation. 1. Original NMR or X-ray 2.After rebuilding side-chains After deleting and rebuilding side-chains 4 get worse > 0.05 nm 7 get better > 0.05 nm Dimer