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Getting the structure of Alyteserin-1a

Alyteserin-1a is a modified antimicrobial peptide which was made by introducing two mutations (D4E & N23S) in Alyteserin-1c peptide. The structure for Alyteserin-1a is not available therefore we obtained the pdb structure for Alyteserin-1c (pdb id : 2L5R) and created the given mutations in pymol. We carefully analyzed the structural feature of Alyteserin-1a peptide to come up with another peptide which could interact with it. We seem to find an interesting pattern which was a hydrophobic groove on one side of the peptide. We targeted this region to neutralize the anit-microbial peptide.

Predicting the structure of Naly

In our project, a key step was to neutralize the antimicrobial peptide, Alyteserin-1a, so that we can create a stress free environment. For this, we designed a novel peptide of smaller length, which can interact favorably with Alyteserin-1a. We used following step to get the best possible peptide :

• We used pepstr, an online tool, which takes amino-acid sequence as input and gives us possible structures, in pdb form, of the protein.
• After getting several possible structures, we used another online tool, ZDOCK, which docks two molecules and give a score, which tells us about that how favorably peptides are interacting.
• We screened few peptides from above process and proceeded for MD simulations.

Molecular Dynamics Simulation

We performed molecular dynamics simulation experiments to confirm that Naly is able to interact with Alyteserin-1a antimicrobial peptide, hence it can neutralize it's activity to kill the pathogemic bacteria. Following are the steps that we followed in this experiment :

1. Convert both the pdb files to .gro files and get the topology files using pdb2gmx tool. Use GROMOS96 53a6 force field.
2. Change the positions of both the peptides in a way that they are far away and disoriented.
3. Build the .gro file for the complex (both proteins) by merging the above .gro files. Create the .top file for complex both peptides.
4. Define the box for the complex and solvate with water molecules.
5. Add NA+ and CL- atoms to neutralize the charge of whole system. Create the index.ndx file using make_ndx.
6. Prepare the .tpr file for energy minimization step.
7. Steepest descent minimization algorithm was used for a maximum of 50,000 steps.
8. Position restrain the water molecules in complex.top file.
9. NVT equilibrate the system. We used leap-frog integrator, lincs constraint_algorithm, Particle Mesh Ewald for electrostatic interactions, position restrain both the peptides, reference temperature 300K with no pressure couppling for 50,000 max steps.
10. NPT equilibrate the system with Parrinello-Rahman pressure coupling.
11. Run the NPT for a total of five times with position constraint [fcx, fcy, fcz] values 1000, 100, 10, 1, in both posre_.itp file. Use the output of one step as the input for the nnext step.
12. Final production run for 30,000 ps.

Analyzing the md simulation

Once we have run the md simulations, we need to validate the simulation based on several parameters, follow the following protocol to obtain and see the results.

1. Using index.ndx file, remove the water and other atoms i.e. NA and CL from the system.
2. Use the complex.gro file and .xtc file to see the simulation in vmd.
3. Using the .edr file calculate different parameters like potential energy, kinetic energy, total energy, radius of gyration etc. to prove your hypothesis.