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What is p7?
• 63 amino acid peptide from Hepatitis C Virus.
• Viroporin,shown to have channel activity in BLM membrane.
• Channel activity inhibited by amantadine, amilorile, iminosugar derivatives.
• Very limited structural information• Experiments have shown it not to be
involved in RNA replication.
What is molecular dynamics?
• Describe the forces on all atoms: – bonded (bonds, angles,
dihedrals)– non-bonded (van der
Waals, electrostatics)• Describe the initial
atom positions:• Integrate: F = ma (a few
million times…)• Result: positions and
energies of all atoms during a few nanoseconds
Secondary structure prediction of p7
Building atomistic model of p7 using prediction.
Convert atomistic p7 to CG p7
Pre-simulation protocol
P7: A ‘Simple’ Membrane Protein
63 residue channel-forming protein from HCV Consensus topology prediction: 2 TM helices Channel formation due to oligomer
Atomistic vs. Coarse-Grained Simulations
CGing: ~4 atoms → 1 particle (including waters) CG gives ~5x reduction in complexity & ~100x speedup (i.e. from days to
hours per ns)
Atomistic simulations are slow … several weeks per simulation Use coarse-grained methods to speed up simulations
detergent protein
Convert p7 model build from InsightII to CG p7.
CG p7 is centered in a box of
10*10*10. (editconf )
Phospholipids or DPC is added at random positions of the box. ( genbox)
Solvate the box with CG water ( genbox )
Neutralize system charges by addition of CG counter-ion. ( genion )
1) Energy minimization
2) MD (grompp, mdrun )
Simulation protocol
Prediction via CG Simulations: P7
• CG simulations of folding & insertion of 2 helix model into a bilayer• All simulations 5 x 2 µs• 2 TM helix hairpin in POPE/POPC bilayers & in DPC micelles• Incomplete insertion of 2nd helix in POPC bilayers
P7 in POPE:POPC 4:1bilayerP7 in POPE:POPC ( 4:1 ) bilayer P7 in POPE:POPC ( 1: 4 ) bilayer
Conclusions and future directions
• Hairpin and L-shaped structure stable for 2µs in different lipid bilayer compositions.
• Coincide with model proposed by Griffin group.• Need to determine if either structure is in an
metastable state, simulated annealing simulations could be done.
• Simulations with more than one p7 could give insights in its oligomeric state.
• Experimentally, X ray crystallography, CD, NMR to determine its structure.
Acknowledgements
I’d like to express my gratitude towards the following people:
• All members of the Sansom group especially Prof Mark Sansom, Dr Phil Biggin.
• Dr Nicole Zitzmann, Thomas Whitfield from Glycobiology.
