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Introduction

Simulation of Conformational Transitions and Free Energy Calculations of PcrA DNA Helicase Hao Wang Molecular Recognition Group School of Pharmacy University of Nottingham. Introduction. DNA helicases are important enzymes involved in DNA replication, repair, and recombination.

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Introduction

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  1. Simulation of Conformational Transitions and Free Energy Calculations of PcrA DNA HelicaseHao WangMolecular Recognition GroupSchool of PharmacyUniversity of Nottingham

  2. Introduction • DNA helicases are important enzymes involved in DNA replication, repair, and recombination. • DNA helicases are molecular motors to separate the ds-DNA through the conformational changes coursed by the ATP hydrolysis. • PcrA DNA helicase from Bacillus Stearothermophilus was crystallized by Dale Wigley and Panos Soultanas

  3. Substrate Complex Product Complex Substrate Complex Cell, Vol. 97, 75-84, April 2, 1999

  4. Research Aim Substrate complex Product complex ? • Find the Reaction Pathway of the process from the substrate complex to the product complex. • Calculate the free energy profile along the pathway.

  5. Simulation System 637 residues in the protein 15 bases on the short chain of DNA, and 20 bases on the long chain 11439 atoms in protein and DNA The ATP hydrolysis wasn’t included Explicit solvent (TIP3P) 52 Na+ ions were added 25419 water molecules were added 87619 atoms in the system Amber force field 300K 8 CPU years simulation on this project

  6. The computational methods to simulate the conformational changes of known ended system: 1. Optimize the first guessed pathway between two conformations. 2. Use extra constraints to drive the molecule in the direction of the target structure.

  7. End Start

  8. Chain Minimization Path

  9. End Start

  10. Targeted Molecular Dynamics

  11. WHAM Weighted Histogram Analysis Method ( WHAM ) is a method using umbrella sampling to provide a free energy profile along a given reaction coordinate. Umbrella sampling is a well-established free energy technique using an artificial biasing window potential to sample unfavourable areas of conformational space. Usually this biasing is a standard harmonic potential applied to a bond length, angle or dihedral. In this research, the RMSD is used as the biasing. Using the TMD to search a certain RMSD region along the reaction path. Using WHAM program written by Alan Grossfield. Computer Physics Communications 91 (1995) 275-282

  12. End Start

  13. End Start • RMSD to the product complex along the Chain Minimization path • RMSD to the product complex along the TMD path If the two reaction pathway are similar, the RMSD values along the diagonal line should be very small. This picture shows the two methods find a similar path.

  14. Free Energy Profile Targeted MD Chain Minimisation The RMSD to the product complex reduces from 3 to 1.7 The free energy of the product complex is higher than that of the substrate complex The height of free energy barriers are different The scale of the free energy profiles are different The ATP hydrolysis only can offer 7.3kcal/mol energy

  15. Future Work √ ? Substrate Complex Product Complex Substrate Complex • Calculating the Free Energy changes from the product complex to the substrate complex. • Investigating the barriers involved. • Coupling between the ATP hydrolysis and the dynamics.

  16. Acknowledgments • Dr Charlie Laughton and Dr Stephen Doughty • Dr Panos Soultanas • Professor Mark Searle • Everyone in the Molecular Recognition Group Dr. Ian Withers Ms. Verity Hudson Mr. Daniel Warner Ms. Michele Burke Dr. Mark Beardsell Mr. Angelo Pugliese Mr. Supat Jiranusornkul • University of Nottingham and Spirogen Ltd for funding

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