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Investigation of Binding Affinity of hIFN-gamma Mutated Forms with the Rosetta Suite of Programs Damyan Grancharov1, Peicho Petkov1, Elena Lilkova1, Nevena Ilieva2, Leandar Litov1 Faculty of Physics, Sofia University “St. Kliment Ohridski”, Sofia, Bulgaria Institute for Nuclear Research and Nuclear Energy, Bulgarian Academy of Sciences, Sofia, Bulgaria 1 2 Overview hIFN-γ and suppressing its abnormal biological activity Protein docking with Rosetta Algorithm Scoring functions hIFN-γ study with Rosetta Conclusions hIFN-γ Powerful immunomodulator; Autoimmune diseases due to abnormal expression; Suppression of biological activity Competitive binding of the hIFN-γR; Mutated analogs of the native hIFN-γ that: Interrupt the signal transduction pathway; Preserve binding ability; 12 mutated analogs out of 100 were selected; Preservation of 3D structure = preservation of binding ability; Protein docking is needed to test such a hypothesis! Rosetta docking Imitates the binding process in real life; Low resolution search: Centroid representation; 500 random perturbations of the current conformation; Low res scoring function; High resolution calculation: All atom representation; High res scoring function; 50 perturbations of random displacement and side-chain optimization; Minimization; Acceptance via Metropolis criterion; High resolution calculation concept Scoring function (1) Low resolution scoring function: Chemical terms: Spair – accounts for pairwise coupling of amino acids; Senv – accounts for the environment of amino acids; Structural terms: Scontact – “reward” term for VdW attractive interaction; Sbump – “penalty” term for VdW repulsive interaction; Salign – term to account for additional biological information; Scoring functions (2) High resolution scoring function: S – scoring function terms; w – weights. Scoring functions (3) Satr – attractive Van der Waals interaction; Srep – repulsive Van der Waals interaction; Ssol – accounts for the presence of an implicit solvent; Ssasa – accounts for the solvent accessible surface area; Shb – accounts for the presence of hydrogen bonds; Sdun – accounts for rotamer probabilities; Spair – accounts for pairwise coupling of amino acids; Selec – electrostatic interaction, divided into short and long ranged, attractive and repulsive; hIFN-γ study with Rosetta For the sake of testing the algorithm: Native hIFN-γ was used; “Blind prediction” protocol was used: Partners were randomized; 50000 models were generated; Results plotted: Score vs RMSD; Best model passed on to refinement; Refinement run: 1000 model were generated; In the Score vs RMSD plot a “funnel” appeared; Blind prediction results Refinement run results Predicted structure RMSD 0,74 Å Conclusions Close-to-native conformations can be obtained, paying the cost of extensive search: Native hIFN-γ with receptor complex reproduced within 0,74 Å; All 12 mutated forms will enter only a refinement procedure: To be adjusted within the funnel in free energy space, encountered for the native hIFN-γ; Thank You for Your Attention! Back-up slides Metropolis acceptance criterion Probability to go from state j to state i: Table of weights