Suppose I have a protein X bound with ATP. I want to mutate a residue ALA interacting with ATP into ASN (or any amino acids). Next, I want to know what effect this mutation has on protein-ligand interaction. Does the interaction become strong or weak, etc.
Please guide me on what steps I need to perform for doing mutation and analyzing the results. I can mutate the amino acids using Pymol and I think after performing mutation I need to perform simulation.
First of all perform a comparative flexible docking experiment. Evaluate your case and co relate the results with facts. Experimenting with simulation starts after previous studies results i.e. Docking (preferably single residue flexible in your case). Check your case if it is a induced fit case then even the dual phase flexible docking will not answer your case properly. Moving ahead in simulations blindly will lead you towards a dark path.
Dear Harinder,
I suggest following steps:
First docking ATP to the mutate pro then performing MD simulation for both natural and mutate structures with same conditions(personally I suggest amber package for md simulation) and finally analyzing trajectories(obtaining H-bonds and binding free energy for both complexes).
Hope it helps,
HHA
I have a question, My PDB is already solved with the given ligand. If i mutate a amino acids interacting with ligands and simulate it. Wouldn't the simulation provide me the information, what happens to interaction between ligand and amino acids. Is it really neccessary to dock the already bound ligand in PDB.
Hi Harinder,
If the ligand is already present inside the enzyme pocket you don't need to dock it.
Just simulate the wild type and the mutant for a long time span and analyze the trajectories.
Ok. Now, my question is how long the simulation time should be, as there is a single mutation in the protein. Should it be 100ps, 1ns etc.
Sometimes changing a simple residue (such as alanine) to bigger one creates overlaps in your pdb file.Check this with a graphical program(e.g VMD).
Base on recent accepted papers simulation period should be between 10 to 50 ns but there is no limitation for it.The more the better
Best
I think after point mutation you need to optimize the protein structure using simulation tools after then use any docking tools to see the effect of mutation on interaction with ATP molecule.
for docking : Autodock tools, Dock etc.
Simulation: GROMACS, AMBER etc.
Visualization: PyMol, Deep View etc.
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