Coarse grained simulations of p7 folding

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 u 63 residue channel-forming protein from HCV u Consensus topology prediction: 2 TM helices u Channel formation due to oligomer

Atomistic vs. Coarse-Grained Simulations u CGing: ~4 atoms → 1 particle (including waters) u CG gives ~5x reduction in complexity & ~100x speedup (i.e. from days to hours per ns) u Atomistic simulations are slow … several weeks per simulation u Use coarse-grained methods to speed up simulations detergentprotein

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 2 nd helix in POPC bilayers P7 in POPE:POPC 4:1bilayer P7 in POPE:POPC ( 4:1 ) bilayerP7 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.