BIOINFORMATICS PROJECT

BY: MIT KOTECHA

CONTENTS.
Aim Introduction Softwares used for modeling Sequence alignment & homology modeling Validation of model Docking Docking Results Conclusions

 Structure

prediction of chitinase(enzyme that degrades chitin layers) isolated from the pathogen Aphanocladium Album ,using web based bioinformatics tools ,molecular modeling softwares and servers. To find effective inhibitor for this enzyme by docking.

INTRODUCTION
Degrades chitin layer by breaking down the glycosidic

bonds. Mostly used for digestion purposes, this enzyme is found in a wide category of organisms including organisms as primitive as bacteria & fungi and as advanced as human beings. Most often the chitinase in pathogens falls under family 18.

 Clinical references associate chitinase with various

allergies (both in animals and plants )including asthma for which exposure to high levels of chitinase is observed to be a cause (though the reason for infection is not clear yet. In microorganisms, chitinase is found as to be a primary metabolite , hence inhibiting chitinase will inhibit the growth of the microorganisms. Therefore we can form antibiotic drugs via inhibiting chitinase.

Pathogen Activity of the Given Fungus

Aphanocladium Album ,though a weak pathogen, is associated with the brown spots growing on the caps of mushrooms. And so this particular fungus is found to be pathogen of another fungus.

SOFTWARES
Modeling involves sequence alignment (single or multiple depending on the requirements) followed by homology modelling or threading(2 structure prediction). From sequence alignment (BLAST) we can find out whether or not the target sequence has a crystal structure assigned in PDB.

 Homology Modeling (automated)can be done by SWISS MODEL, PHYRE, GENO3D . The acquired models are to be verified for validation. This can be done either by VERIFY-3D or by Ramachandran plot (wincoot).

Sequence alignment and homology modelling

Sequence of the query protein obtained from protein database of NCBI( www.ncbi.nlm.nih.gov) . This sequence is put into BLAST programme of NCBI and the closest homologue is determined to be crystal structure of a chitinase CrChi1 from the nematophagous fungus Clonostachys rosea( pdb code 3G6L).

BLAST indicates 2 things. 1.The target sequence doesnt have a known structure. 2.For the homology modelling ,if to be done manually , the protein with max sequence identity(70%). After BLAST we need to do the homology modelling. As mentioned earlier the automated homology modeling was performed by using three programs: SWISS MODEL, PHYRE, and GENO3D.

The validation for the structure models obtained from various software tools (SWISS MODEL, PHYRE, GENO3D), was done by analyzing the psi-phi Ramachandran plot generated by wincoot. The compatibility of the predicted structures with the given sequence was also verified using VERIFY-3D. The entire process of verification pointed towards the structure predicted by swiss-model.

DOCKING
Molecular docking is a widely-used computational tool for the study of molecular recognition, which aims to predict the binding mode and binding affinity of a complex formed by two or more constituent molecules with known structures. An important type of molecular docking is protein-ligand docking because of its therapeutic applications in modern structure-based drug design.

 Inhibitor is a substance that inhibits the action of an enzyme. By finding inhibitors to the given protein and making a protein- inhibitor complex helps a lot in antibiotic drug preparation. To calculate the exact position where the inhibitor should bind ,having minimum energy ineractions , we use computational docking. For the protein of interest , the suitable ligands are found to be methyl xanthine derivatives (Caffeine, Threophylline) & allosamidin.

We docked three different ligands with the obtained 3D structure of the given protein using Hex. This was done to find the energy interactions between the ligands and the given protein. The functions performed by Hex in order to find the best possible protein-ligand interaction are: SPF Transform, FFT streric scan ,FFT Final Search , MM Refinement, Total Dockings.

DOCKING RESULTS

DOCKING RESULTS (contd)

Energy Interactions of Each Complex

DOCKING CONCLUSIONS
The comparision of the total energies of interaction shows

that the best inhibitor(ligand) for the given protein molecule is Allosamidin. Allosamidin has the minimum amount of interaction energy ,i.e. the interaction between the given protien and allosamidin is most feasible as compared to other ligands docked. Hence ,out of the given 3 molecules, Allosamidin turns out to be the most potent inhibitor. APPLICATION :This process is used to make Anti-fungal drugs these days by inhibiting the chitinase function in fungi.