1. Human Pancreatic Ribonuclease Molecular Simulations to compare it with Ribonuclease A Group Meeting Xavier Prat-Resina April 20 th 2007
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4. 1. Introduction: Struct. & React. Structure: HP-RNase with tetra-nucleotide (4 cytidine) in endo coordination The difference between HP-Rnase and Rnase A is that the former has 2 more positively charged residues at the N-terminal: Lys6 and Arg4
5. 1. Introduction: Struct. & React. Enzymatic reaction The substrate for our simulations: tetra-cytidilic acid with a cyclic phosphate
6. 1. Introduction: Struct. & React. Active site and binding sites in Exo coordination 4-Cytidine with cyclic phosphate Endo/Exo P3/P2 P2/P1 P1/P0 P2/P-1 Exo product Endo Product
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10. 3. Setup for Simulations Homology modelling (Alex Gomez-Garrido): Initial structures: RNAse A bovine 1RCN and HP-RNase mutant 1DZA with 4 DNA nucleosides (ATAA) The structure are all very similar
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13. Analysis of important distances We want to see the role of different residues Differences between exo/endo and wild type and mutant 4. Struct. Analysis of MD Lys1 Arg4 Lys7 Arg10 Lys6
14. Arg4-P2 endo exo Arg4-P3 4. Struct. Analysis of MD Analysis of important distances
15. Lys6-P2 endo exo Lys6-P3 4. Struct. Analysis of MD Analysis of important distances
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17. D Gtot(Wild type) > D Gtot(mutant) ? The ratio between exo and endonucleolytic product is 1.35 in HP-RNase and 2.15 for the mutant. D Gtot ? Endo Exo 5. Free Energy Calculations D G1 D G2 D Gtot= D G 1 + D G2 Alchemical pathway
18. Alchemical Free Energy 5. Free Energy Calculations We run Molecular Dynamics and collect the energy difference between reactant ”a” and product ”b” To converge the calculation we alchemically transform ”a” into ”b” scaling the two structures. We define a system as a combination of reactants ”a” and products ”b” We run several ”windows” at different values of l and combine them (100 ps/window)
19. 5. Free Energy Calculations D G1 D G2 Wild Type Mutant Free Energy (Kcal/mol) Lambda (0-1)
20. D G 1=202.54 D G 2=-199.17 D G tot= 202.54 -199.17 =3.37 kcal/mol Wild Type Endo Exo 5. Free Energy Calculations D G tot= 198.90-189.27 =8.63 kcal/mol D G 1=198.90 D G 2=-189.27 Mutant
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