TRAINING REPORT ON INSILICO STUDY OF MACROMOLECULES

Submitted To:
Mr. Siddharth Sinha

Submitted By:
Pragya Pandey

ACKNOWLEDGEMENT:
I have taken efforts in this training. However, it would not have been possible without the kind support and help of many individuals and organizations. I would like to extend my sincere thanks to all of them. I am highly indebted to Mr. Siddharth Sinha for his guidance and constant supervision as well as for providing necessary information regarding the training & also for their support in completing the training. I would like to express my gratitude towards my parents & my brother for their kind cooperation and encouragement which help me in completion of this training. My thanks and appreciations also go to my colleague in training and people who have willingly helped me out with their abilities.

INTRODUCTION:
Herpes zoster, commonly known as Shingles, is a viral disease characterized by painful skin rash with blisters in a limited area on one side of body, often in a stripe. This disease is
caused by Vericella zoster virus. Primary VZV results in chicken pox. Even when clinical symptom of chickenpox have resolved, VZV remain dormant in nervous system of infected person(virus latency) in the trigeminal and dorsal root ganglia. In about 10-20% of cases, VZV reactivates later in life producing disease shingles.VZV is commonly known as Human Herpes Virus Type3. Shingles rash starts with small blisters on red base, with new blisters countinuing to form for three to five days. Blisters follow the path of individual nerves that come out of the spinal cord in a specific "ray like" distribution (called a dermatomal pattern) and appear in a band like on an area of skin. The entire path of the affected nerve may be involved or there may be areas in the distribution of nerve with blisters or areas without blisters. Evetually the blisters pop, and the

area starts to ooze. The affected areas wil then crust over and heal. The duration of outbreak may take three to four weeks from start to finish. On occasion the pain will be present, but the blisters may never appear.

The enveloped virus creates the chickenpox rash and can travel from the skin to sensory nerves. Once in the sensory nerves, the virus moves to the sensory ganglia where it becomes latent. If reactivated, the virus travels from the sensory ganglia back to the skin where it creates the shingles rash.

Proteins are an important class of biological macromolecules present in all organisms. All proteins are polymers of amino acids. Classified by their physical size, proteins are nanoparticles (definition: 1 100 nm). Each protein polymer also known as a polypeptide consists of a sequence of 20 different L- -amino acids, also referred to as residues. For chains under 40 residues the term peptide is frequently used instead of protein. To be able to perform their biological function, proteins fold into one or more specific spatial conformations, driven by a number of non-covalent interactions such as hydrogen bonding, ionic interactions, Van Der Waals forces, and hydrophobic packing. To understand the functions of proteins at a molecular level, it is often necessary to

determine their three-dimensional structure. This is the topic of the scientific field of structural biology, which employs techniques such as X-ray crystallography, NMR spectroscopy, and dual polarisation interferometry to determine the structure of proteins. A ligand (from the Latin ligandum, binding) is a substance that forms a complex with a biomolecule to serve a biological purpose. In a narrower sense, it is a signal triggering molecule, binding to a site on a target protein. The binding occurs by intermolecular forces, such as ionic bonds, hydrogen bonds and van der Waals forces. The docking (association) is usually reversible (dissociation). Actual irreversible covalent binding between a ligand and its target molecule is rare in biological systems. Ligand binding to a receptor alters the chemical conformation, which is the three dimensional shape of receptor protein. The conformational state of a receptor protein determines the functional state of a receptor. Ligands include substrates, inhibitors, activators, and neurotransmitters. In this report the stucture of protein and ligand for drug designing, procedure of

prediction of protein structure i.e. homology modelling, interaction between protein and ligand i.e. molecular docking and calculation of the time dependent behavior of a molecular system i.e. molecular dynamics has been described.

METHOS AND MATERIAL:

First of all we identify potential target for Herpes zoster. TARGET IDENTIFICATION For the pharmaceutical industry, the discovery of a new drug presents a enormous scientific challenge and consists essentially in the identification of new molecules or compounds ideally, the latter will become new drugs that act in new ways upon biological targets specific to the disease requiring therapeutic approaches. The indentification of therapeutic targets requires

knowledge of disease 's etiology and the biological systems associated with it. Molecular biology has revlutionized the process of drug discovery. Not too long ago, scientists searched for new targets employing a long and costly process of trial and error. Today, the collective contribution of genomics, proteomics and bioinformatics allows for the much more rapid and precise discovery of those genes and/or proteins involved in the etiology of certain disease.

The simplest method of identification relies on serial pair wise sequence alignments aided by databases search techniques such as FASTA and BLAST. This family of methods has been shown to produce a larger number of potential templates and to identify better templates for sequences that have only distant relationships to any solved structure. When performing a

BLAST search, a reliable first approach is to identify hits with a sufficiently low E-value, which are considered sufficiently close in evolution to make a reliable homology model.

DATABASES: NCBI www.ncbi.nlm.nih.gov/ PDB www.rcsb.org/ MSD www.rcsb.org/databases.html CATH www.biochem.ucl.ac.uk/bsm/cath/ TrEMBL srs.ebi.ac.uk/ Scop scop.mrc-lmb.cam.ac.uk/scop/ GenBank

www.ncbi.nlm.nih.gov/Genbank/GenbankSearch.html PSI www.structuralgenomics.org

SOFTWARE TOOLS: BLAST http://www.ncbi.nlm.nih.gov.library.vu.edu.au/BLAST

Target Identification: Target for Herpes zoster disease can be identified using literature review. After reviewing the Literature, a novel target was identified and Information like name and origin, entry information, references, cross references, databases and sequence of this target was retrieved in UniProtKB/TrEMBL, a computer-annotated supplement of SwissProt that contains all the translations of EMBL nucleotide sequence entries not yet integrated in Swiss-Prot. Name of Target: Transcriptional regulator IE63(ICP22)

Template identification Once the target was identified, template identification relies on serial pair wise sequence alignments aided by database search techniques such as FASTA and BLAST.

HOMOLOGY MODELLING Now we construct an atomic resolution model of "target" protein by its amino acid saquence and an experimental three-dimensional structure of a related homologous protein (the "template"). It is done by two methods of Homology Modelling i.e. SwissPdb viewer and Modeller. Homology modeling relies on the identification of one

or more known protein structures likely to resemble the structure of the query

sequence, and on the production of an alignment that maps residues in the query sequence to residues in the template sequence. It has been shown that protein structures are more conserved than protein sequences amongst homologues, but sequences falling below a 20% sequence identity can have very different structure.

i.PROTEIN MODELLING USING SPDBV: Swiss-Pdb Viewer (or SPDBV), is an interactive molecular graphics program for viewing and analyzing protein and nucleic acid structures. In combination with Swiss-Model (a server for automated comparative protein modeling maintained at http://www.expasy.org/swissmod) new protein structures can also be modeled. A molecular coordinate file (e.g. *.pdb.) is a text file containing, amongst other information, the atom coordinates of one or several molecules. It can be opened from a local directory or imported from a remote server by entering its PDB accession code. The content of one coordinate file is loaded in one (or more) layers, the first one will be referred to as the "reference layer". Deep View can simultaneously display several layers, and this constitutes a project. When working on projects, the layer that is currently governed by the Control Panel is called the currently active layer. Each molecule is composed of groups, which can be amino acids, hetero-groups, water molecules, etc. and each group is composed of atoms. Non-coordinate files containing specific information other than atom coordinates. Molecular surfaces, electrostatic potential maps, and electron density maps are examples of non-coordinate files, which can either be computed by DeepView, or loaded from specialized external programs. PROCEDURE: Protein sequence accession number: PO8354 Swiss-prot protein entry: GE_SUHVR The protein sequence from Swissprot GE_ SUHVR was downloaded and saved in FASTA format. BLAST search was performed to get the template for modeling.  2AWH/ A was selected as template which showed around 34% identity with target

sequence and the template structure was downloaded from the PDB.  Swiss-Pdb Viewer was launched and the following procedure was carried out. Steps involved in SPDBV:  open the template structure from file (.pdb file)  select the A chain  choose icon -'select'-'inverse selection'  choose icon -'build' -'removed selected residues'  choose icon -'file' - 'save'-'layer' (".pdb")  choose icon - 'Swiss model'-'load the raw target sequence  choose icon -'fit'-'fit raw sequence' then 'magic fit' then 'iterative fit'  choose icon -'file' - 'save'-'layer'(".pdb")  choose icon -'file' - 'save'-'project'(".pdb")  choose icon - 'Swiss model'-'submit modeling request'(A new browser will be opened loading the pdb file and give the Email ID for receiving the modeled structure)  open the new structure (received from Email) - remove the template-by selecting the target.  choose icon -'file' - 'save'-'layer'(".pdb")

  Open Swiss model and select load raw sequence option to load target molecule.

 Perform magic fit, iterative fit provided under FIT in order to fit the two sequences.

 Save the file as the project

 Select submit modeling request under Swiss model to submit it for modeling.

  The project comes back at the e-mail address you have provided.

 In the control panel present in WINDOWS, select only the target sequence and delete the template. Hence we will be left with modeled structure of our target protein.

Result Thus the final protein is obtained from the SPDBVserver and this can be used for further Analysis. ii. PROTEIN MODELING USING MODELER:

Modeller is a computer program that models three-dimensional structures of protein and their assemblies by satisfaction of spatial restraints. It is most frequently used for homology or comparative protein structure modeling: The user provides an alignment of a sequence to be modeled with known related structures and MODELLER will automatically calculate a model with all non-hydrogen atoms. It can also perform

multiple comparison of protein sequences and/or structures, clustering of proteins, and searching of sequence databases. The program is used with a scripting language and does not include any graphics. It is written in standard FORTRAN 90 and will run on UNIX, Windows, or Mac computers.

Comparative protein structure modeling by MODELLER MODELLER implements an automated approach for comparative protein structure modeling. The input are from the Protein Data Bank (PDB) atom files of known protein structures, and their alignment with the target sequence to be modeled, and the output is a model for the target that includes all non-hydrogen atoms. Although MODELLER can find template structures as well as calculate sequence and structure alignments, it is better in the difficult cases to identify the templates and prepare the alignment carefully by other means. The alignment can also contain very short segments such as loops, secondary structure motifs, etc. Given an alignment, the model is obtained without any user intervention.

Target sequence
>sp|P08354|GE_SUHVR Envelope glycoprotein E OS=Suid herpesvirus 1 (strain Rice) GN=gE PE=3 SV=1MRPFLLRAAQLLALLALALSTEAPSLSAETTPGPVTEVPSPSAEVWDLSTEAGDDDLDGDLNGDDRRAGFGSALASLREAPPA HLVNVSEGANFTLDARGDGAVVAGIWTFLPVRGCDAVAVTMVCFETACHPDLVLGRACVPEAPERGIGDYLPPEVPRLQREPPIVT PERWSPHLTVRRATPNDTGLYTLHDASGPRAVFFVAVGDRPPAPLAPVGPARHEPRFHALGFHSQLFSPGDTFDLMPRVVSDMGD SRENFTATLDWYYARAPPRCLLYYVYEPCIYHPRAPECLRPVDPACSFTSPARAALVARRAYASCSPLLGDRWLTACPFDAFGEEVHT NATADESGLYVLVMTHNGHVATWDYTLVATAAEYVTVIKELTAPARAPGTPWGPGGGDDAIYVDGVTTPAPPARPWNPYGRTTP GRLFVLALGSFVMTCVVGGAVWLCVLCSRRRAASRPFRVPTRAGTRMLSPVYTSLPTHEDYYDGDDDDEEAGDARRRPSSPGGDS GYEGPYVSLDAEDEFSSDEDDGLYVRPEEAPRSGFDVWFRDPEKPEVTNGPNYGVTASRLLNARPA

Template sequence
>2AWH DLKAFSKHIYNAYLKNFNMTKKKARSILTHTAPFVIHDIETLWQAEKGLVWKEISVHVFYRCQCTTVETVRELTEFAKSIPSFSSLFLND QVTLLKYGVHEAIFAMLASIVNKDGLLVANGSGFVTREFLRSLRKPFSDIIEPKFEFAVKFNALELDDSDLALFIAAIILCGDRPGLMNVP RVEAIQDTILRALEFHLQANHPDAQQLFPKLLQKMADLRQLVTEHAQMMQRIKKTETETSLHPLLQEIYKDMY

Preparing input files The sample input files in this found in the directory of the MODELLER distribution. There are three kinds of input files: Protein Data Bank atom files with coordinates for the template structures, the alignment file with the alignment of the template

structures with the target sequence, and MODELLER commands in script files that instruct MODELLER what to do.

Atom files Each atom file is named code.atm where code is a short protein code, preferably the PDB code;, Lipopolysaccharide heptosyltransferase 1.be in a file chain_a.atm. The code must be used as that protein's identifier throughout the modeling.

ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

N CA C O CB CG

ASP A 210 ASP A 210 ASP A 210 ASP A 210 ASP A 210 ASP A 210

19.517 -22.627 20.002 -21.881 19.030 -20.740 19.046 -19.653 21.418 -21.354 22.002 -20.621 23.126 -20.082 21.347 -20.575 18.183 -21.011 17.121 -20.103 17.695 -18.830 17.021 -17.807 16.219 -20.815 14.876 -21.429 14.737 -21.784 14.562 -22.613 18.951 -18.898 19.598 -17.768 20.184 -16.802 20.111 -15.576 20.645 -18.291 21.422 -17.239 22.502 -17.891 23.277 -16.857 23.603 -17.337 20.752 -17.374 21.241 -16.625 20.105 -15.950 20.227 -14.812 22.007 -17.553

41.103 42.291 42.653 42.051 42.030 43.218 43.074 44.286 43.639 44.047 44.658 44.725 45.046 44.619 43.110 45.515 45.096 45.766 44.721 44.873 46.767 47.537 48.407 49.208 50.610 43.657 42.499 41.714 41.255 41.572

1.00 46.23 1.00 46.53 1.00 45.73 1.00 46.00 1.00 46.93 1.00 48.44 1.00 50.56 1.00 49.05 1.00 44.68 1.00 42.93 1.00 42.05 1.00 41.74 1.00 43.18 1.00 43.39 1.00 42.62 1.00 42.95 1.00 40.71 1.00 40.18 1.00 38.47 1.00 38.15 1.00 39.80 1.00 41.95 1.00 42.39 1.00 46.53 1.00 48.80 1.00 36.73 1.00 34.74 1.00 33.04 1.00 31.56 1.00 34.74

OD1 ASP A 210 OD2 ASP A 210 N CA C O CB CG LEU A 211 LEU A 211 LEU A 211 LEU A 211 LEU A 211 LEU A 211

CD1 LEU A 211 CD2 LEU A 211 N CA C O CB CG LYS A 212 LYS A 212 LYS A 212 LYS A 212 LYS A 212 LYS A 212

CD LYS A 212 CE NZ N CA C O CB LYS A 212 LYS A 212 ALA A 213 ALA A 213 ALA A 213 ALA A 213 ALA A 213

ALIGNMENT
>P1;2AWH structureX:2AWH:210 :A:477 :A:ferredoxin:Azotobacter vinelandii: 1.90: 0.19 ---------------------DLKAFSKHIYNAYLKNFNMTKKKARSILT HTAPFVIHDIETLWQAEKGLVWKEISVHVFYRCQCTTVETVRELTEFAKS -------IPSFSSLFLNDQVTLLKYGVHEAIFAMLASIVNKDGLLVANGS G-FVTREFLRSLRKPFSDIIEPKFEFAVKFNALELDDSDLALFIAA-----IILCGDRPGLMNVPRVEAIQDTILRALEFHLQANHP-DAQQLFPKLLQ KMADLRQLVTE-----HAQMMQR--IKKTETETSLHPLLQEIYKDMY----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------* >P1;target sequence:target:1 : :577 : :ferredoxin:Peptococcus aerogenes: 2.00:-1.00 MRPFLLRAAQLLALLALALSTEAPSLSAETTPGPVTEVPSPSAEVWDLST EAGDDDLDGDLNGDDRRAGFGSALASLREAPPAHLVNVSEGANFTLDARG DGAVVAGIWTFLPVRGCDAVAVTMVCFETACHPDLVLGRACVPEAPERGI GDYLPPEVPRLQREPPIVTPERWSPHLTVRRATPNDTGLYTLHDASGPRA VFFVAVGDRPPAPLAPVGPARHEPRFHALGFHSQLFSPGDTFDLMPRVVS DMGDSRENFTATLDWYYARAPPRCLLYYVYEPCIYHPRAPECLRPVDPAC SFTSPARAALVARRAYASCSPLLGDRWLTACPFDAFGEEVHTNATADESG LYVLVMTHNGHVATWDYTLVATAAEYVTVIKELTAPARAPGTPWGPGGGD DAIYVDGVTTPAPPARPWNPYGRTTPGRLFVLALGSFVMTCVVGGAVWLC VLCSRRRAASRPFRVPTRAGTRMLSPVYTSLPTHEDYYDGDDDDEEAGDA RRRPSSPGGDSGYEGPYVSLDAEDEFSSDEDDGLYVRPEEAPRSGFDVWF RDPEKPEVTNGPNYGVTASRLLNARPA*

Script fileMODELLER is a command-line only tool, and has no graphical user interface; instead, you must provide it with a script file containing MODELLER commands. This is an ordinary Python script.

Running MODELLER To run MODELLER with the script file script.py above, we do the following: 1. Open a command line prompt: o On Windows: Click on the `Modeller' link on Start Menu. This will give a Windows Command Prompt, set up for to run MODELLER.

2. Change to the directory containing the script and alignment files created earlier, using the 'cd' command. 3. Run MODELLER itself by typing the following at the command prompt:
mod9v2 model.py

A number of intermediary files are created as the program proceeds. After about 10 seconds on a modern PC, the final chain_a model is written to file P2O4H.B99990002.pdb. Examine the script.log file for information about the run.

Thus the final protein is obtained from the MODELLER server and this can be used for further Analysis.

Model Validation We validate the structure of target protein ICP22 by determining the overall quality factor and Ramachandran plot value using SAVS. Structure Analysis and Validation Server greatly simplifies computational analysis of the molecular structure and sequence of proteins. The stereochemical validation of model structures of proteins is an important part of the comparative molecular modeling process. Ramachandran plot is a way to visualize dihedral angles

against

of amino acid residues in protein structure. It shows the possible and angles for a polypeptide. The Ramachandran plot displays the

conformations of

psi and phi backbone conformational angles for each residue in a protein. The distance between two succession alpha carbon atoms in the backbone chain and the angles between the two bonds of such atoms in desired protein can be determined using this plot. Software SAVS: http://nihserver.mbi.ucla.edu/SAVS/ Procedure The target protein structure ICP22 obtained after homology modeling using deep view and modeller is given as input for SAVS.

SAVS Output: SAVS for modeled using deep view Quality Factor-96.141

SAVS for modeled using MODELLER Quality Factor-94.855

The SAVS output for model predicted using MODELLER is better compared to that of Deep View is inferred based on its Ramachandran plot value.

LIGAND IDENTIFICATION Then we find one or more ligands that interacts with our Target protein. Once the therapeutic target has been identified, we must then find one or more leads (e.g., chemical compounds or molecules) that interact with the therapeutic target so as to induce the desired therapeutic effect, e.g., through antiviral or antibacterial activity. In order to discover the compounds whose pharmacological properties are likely to have the required therapeutic effects, researchers must test a large variety of them on one or more targets. The pharmaceutical companies possess veritable libraries of synthetic or natural compounds, ready to be tested. To test the chosen compounds in large numbers, scientists use an entirely automated process known as high density screening. In general, of the thousands of compounds tested, barely 1% will qualify for further and more probing analysis. Databases NCI Pubchem ZINC Tools: To convert mol2 file to pdb file we use World Wide Molecular Matrix http://wwmm-svc.ch.cam.ac.uk/wwmm/html/observer.html http://129.43.27.140/ncidb2/ http://pubchem.ncbi.nlm.nih.gov/ http://zinc.docking.org/

Procedure There are two ways to find Lead compounds that interact with our Target protein.  Using Literature Survey  Comparative analysis, finding ligand that binds to template protein and use that ligand and its similar ligand.

Using Literature Survey After a thorough literature survey, it was found that Roxytal is one of the potential drugs used against HERPES ZOSTER. So a zinc database compound search was done and Roxytal was selected as one of the leads.

Comparative Analysis In the PDB page of 2AWH (template) the ligand compounds that interact with it are identified. Using a zinc database compound search similar compounds to that ligand were found and included as leads.

Conversion of mol files to Pdb The structure of the ligand got from Zinc database was in mol2 format. These structure files were converted to Pdb file using World wide molecular matrix (Open Babel) tool.

The MOL2 file is opened in a word pad and its content is copied and given as the input to open Babel server (Input format mol, Output format pdb). The result from the server is pasted in a word pad and saved as a .pdb file. Thus lead compounds that interact with our target protein molecule were identified with help of Zinc database.

MOLECULAR DOCKING Then we predict the affinity, activity, binding orientation of our ligand Vaccenic Acid to our target proteinICP22 , i.e. to perform docking using AUTODOCK. Docking is a method which predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. Knowledge of the preferred orientation in turn may be used to predict the strength of association or binding affinity between two molecules using for example scoring functions. The associations between biologically relevant molecules such as proteins, nucleic acids, carbohydrates, and lipids play a central role in signal transduction. Furthermore, the relative orientation of the two interacting partners may affect the type of signal produced (e.g., agonism vs. antagonism). Therefore docking is useful for predicting both the strength and type of signal produced. Docking is frequently used to predict the binding orientation of small molecule drugcandidates to their protein targets in order to in turn predict the affinity and activity of the small molecule. Hence docking plays an important role in the rational design of drug. Given the biological and pharmaceutical significance of molecular docking, considerable efforts have been directed towards improving the methods used to predict docking. Auto Dock is a suite of automated docking tools. It is designed to predict how small molecules, such as substrates or drug candidates, bind to a receptor of known 3D structure. Auto Dock actually consists of two main programs: AutoDock performs the docking of the ligand to a set of grids describing the target protein; Auto Grid precalculates these grids. In addition to using them for docking, the atomic affinity grids can be visualized. This can help, for example, to guide organic synthetic chemists design better binders. Also there is a graphical user interface called AutoDock Tools, or ADT for short, which amongst other things helps to set up which bonds will treated as rotatable in the ligand and to analyze dockings.

AutoDock has applications in: y y y y y y y X-ray crystallography; structure-based drug design; lead optimization; virtual screening (HTS); combinatorial library design; protein-protein docking; chemical mechanism studies

SOFTWARE AutoDock : PROCEDURE: Steps involved in AutoDock Initially save the protein and the ligand molecule as protein.pdb and ligand.pdb in a new folder in the desktop. Here we do AutoDock4 in Windows. http://autodock.scripps.edu/

 Open terminal and give in the following commands y cd Desktop ycd New Folder   Prepare protein molecule y y y y   Prepare Ligand molecule y y y Ligand----Input----Open (ligand.pdb) Ligand----Torsion tree----Detect root. Ligand-----Output----Save as PDBQT (ligand.pdbqt) File----Read Molecule----Open (protein.pdb). Edit-----Hydrogens----Add----Polar Only. Save-----Write PDBQT-----(Add ATOM, CONNECT, TER, END and select sort nodes and save transformed coordinates) OK.

 Set Grid Box y y y Grid-----Macromolecule----Choose-----Protein Save as protein.pdbqt. Grid-----Grid Box (Select the three coordinates such that the active site is

covered in the Grid map. Note that three coordinates should be equal) , Click centre pick an atom to place our active site as centre of grid. Go to file in grid box then close saving current. y y Grid-----Output-----Save(protein.gpf) (To display label of active site) Display residue name.  Running the Grid y Run-----Run Auto grid(give in the program path for the auto grid file and the .gpf file) y Wait until the Successful completion of Auto grid is displayed in the terminal.  Docking y y y   Run Docking y Run-----Run Auto Dock (Give in the pathname for Auto Dock and Dpf file). y   Commands for Auto Dock & Auto grid are given in Cygwin as shownWait until successful completion of Docking. Docking-----Macromolecule-----Choose rigid molecule-----rigid.pdbqt. Docking-----Ligand-----Choose-----Select ligand-----Accept. Docking-----Output-----Lamarckian GA-----Save dpf(protein.dpf) label-----by properties-----label only

Output The above steps were performed using our ligand Vaccenic Acid to our target protein ICP22and we got 10 conformations out of which the binding energy was found to be lowest in the 1th conformation. The binding energy was found to be +18.41.

CLUSTERING HISTOGRAM ____________________ ________________________________________________________________ ________________ | | | | | Clus | Lowest | Run | Mean | Num | Histogram -ter | Binding | | Binding | in | Rank | Energy | | Energ | Clus| 5 10 15 20 25 30 35 _____|__________|____ |________ _|_____|____:____|____:____|____:____|____:___ 1 | +18.41 | 4 | +24.79 | 2 |## 2 | +22.25 | 6 | +22.25 | 1 |# 3 | +22.72 | 10 | +22.72 | 1 |# 4 | +23.16 | 3 | +23.16 | 1 |# 5 | +24.77 | 2 | +24.77 | 1 |# 6 | +25.97 | 5 | +25.97 | 1 |# 7 | +26.32 | 9 | +26.32 | 1 |# 8 | +28.41 | 8 | +28.41 | 1 |# 9 | +30.29 | 1 | +30.29 | 1 |# _____|__________|_____|__________|_____|________________________ ______________ Number of multi-member conformational clusters found = 1, out of 10 runs. Chimera is a highly extensible program for interactive visualization and analysis of molecular structures and related data, including density maps, supramolecular assemblies, sequence alignments, docking results, trajectories, and conformational ensembles. High-quality images and animations can be generated. Chimera includes complete documentation and several tutorials, and can be downloaded free of charge for academic, government, non-profit, and personal use.

The interaction between ligand and protein shown in Lines

The Interaction between the ligand and Protein shown in Chimera model

The interaction between ligand and protein shown in Lines

The interaction between the protein and ligand shown in Ribbon model

Thus docking was done for our ligand Vaccenic Acid to our target protein ICP22, using Auto Dock and the binding energy was found to be highest in the 1 st conformation. The binding energy came out to be +18.41.

MOLECULAR DYNAMICS

Molecular dynamics (MD) is a computer simulation of physical movements of atoms and molecules. MD simulations have provided detailed information on the fluctuations and conformational changes of proteins and nucleic acids. The atoms and molecules are allowed to interact for a period of time, giving a view of the motion of the atoms. In the most common version, the trajectories of molecules and atoms are determined by numerically solving the Newton's equations of motion for a system of interacting particles, where forces between the particles and potential energy are defined by molecular mechanics force fields.They are also used in the determination of structures from x-ray crystallography and from NMR experiments. Because molecular systems consist of a vast number of particles, it is impossible to find the properties of such complex systems analytically; MD simulation circumvents this problem by using numerical methods. However, long MD simulations are mathematically ill-conditioned, generating cumulative errors in numerical integration that can be minimized with proper selection of algorithms and parameters, but not eliminated entirely. The results of molecular dynamics simulations may be used to determine macroscopic thermodynamic properties of the system based on the ergodic hypothesis: the statistical ensemble averages are equal to time averages of the system. MD has also been termed "statistical mechanics by numbers" and "Laplace's vision of Newtonian mechanics" of predicting the future by animating nature's forces and allowing insight into molecular motion on an atomic scale. Firstly we convert mol file of ligand into zip file in PRODRG server. By this we find ligand input file for GROMACS. GROMACS is a versatile package to perform molecular dynamics, i.e. simulate the Newtonian equations of motion for systems with hundreds to millions of particles. It is primarily designed for biochemical molecules like proteins, lipids and nucleic acids that have a lot of complicated bonded interactions, but since GROMACS is extremely fast at calculating the nonbonded interactions (that usually

dominate simulations) many groups are also using it for research on non-biological systems, e.g. polymers. By giving specific commands we get em.log ,pr.log, md.log files. Then we can generate graph of RMSD vs time. y pdb2gmx -ignh -ff gmx -f 2AWH.pdb -o prt.pdb -p prt.top -water spce missing

editconf -bt cubic -f prt.pdb -o prt.pdb -d 0.9

genbox -cp prt.pdb -cs spc216.gro -o prt_b4em.pdb -p prt.top

grompp -f em.mdp -c prt_b4em.pdb -p prt.top -o prt_em.tpr

genion -s prt_em.tpr -o prt_ion.pdb -nname CL -nn 1 -g prt_ion.log

grompp -f em.mdp -c prt_ion.pdb -p prt.top -o prt_em.tpr

nohup mdrun -v -s prt_em.tpr -o prt_em.trr -c prt_b4pr.pdb -e em.edr -g em.log &

grompp -f pr.mdp -c prt_b4pr.pdb -p prt.top -o pr.tpr -maxwarn 3

nohup mdrun -s pr.tpr -o pr.trr -c prt-b4md.pdb -e pr.edr -g pr.log &

grompp -f md.mdp -c prt-b4md.pdb p prt.top o md.tpr maxwarn 3

nohup mdrun s md.tpr o md.trr c pr_md.pdb e md.edr g md.log &

trjconv f md.trr o md.xtc ngmx f md.trr s md.tpr

g rms s md.tpr f md.trr o prt rmsd.xvg

xmgrace nxy prt_rmsd.xvg

From this graph we find that time taken for root mean square deviation is approx. 16 ps.

RESULT AND DISSCUSION:

y HOMOLOGY MODELLING
The Target protein obtained from Modeller & Swiss prot through can be viewed through Ramachandran plot as shown-

From Swiss Prot:

From Modeller:

As, we can see in Ramachandran plot that percentage residues in most favoured regions is 91% in case of modeller. So, for our further studies we choose the target designed by the modeller rather than choosing the target from SPDV i.e. SwissProt. For,further study on protein orientation & to predict the affinity, activity, binding orientation of our ligand Vaccenic Acid to our target protein ICP22, i.e. to perform docking using AUTODOCK.

MOLECULAR DOCKING

Result, that we get are in form of glg & dlg files we can be shown as follows-

CLUSTERING HISTOGRAM ____________________ ________________________________________________________________ ________________ | | | | | Clus | Lowest | Run | Mean | Num | Histogram -ter | Binding | | Binding | in | Rank | Energy | | Energ | Clus| 5 10 15 20 25 30 35 _____|__________|____ |________ _|_____|____:____|____:____|____:____|____:___ 1 | +18.41 | 4 | +24.79 | 2 |## 2 | +22.25 | 6 | +22.25 | 1 |# 3 | +22.72 | 10 | +22.72 | 1 |# 4 | +23.16 | 3 | +23.16 | 1 |# 5 | +24.77 | 2 | +24.77 | 1 |# _____|__________|_____|__________|_____|________________________ Number of multi-member conformational clusters found = 1,out of 10 runs. As, we see that the docking energy is found to be +24.79 in the 1 st conformation, so that ligand is selected.

Now,we open the choosen ligand pdb in chimera & visualize the results, as shownFirst,we open the pdb in chimera-

Then, we select the B chain & hide it.

Now,we select the desired residue in remaining A chains.

In modeling that the structures obtained from the deep viewer (spdv) do not have all the amino acids in the preferred regions as in case of modeler which have all the amino acids in the preferred regions, the visualization of the preferred protein is very Important aspect in drug designing, one of the method is auto dock in which we visualize the glg & dlg files for the minimum binding energy which have been given in the reportAs per the binding energy and log P the solubility of the ligand structure is rated. The structure having best solubility ratio is chosen and visualized in Chimera. Chimera is a highly extensible program for interactive visualization and analysis of molecular structures and related data, including density maps, supramolecular assemblies, sequence alignments, docking results, trajectories, and conformational ensembles. High-quality images and animations can be generated. Chimera includes complete

documentation and several tutorials, and can be downloaded free of charge for academic, government, non-profit, and personal use. The Chimera extension view dock facilitates interactive selection of promising compounds from the output of dock. The molecules can be viewed in the context of the binding site and optionally, screened by number of hydrogen bonds to the receptor. In chimera we open our protein selected and superimpose with the target designed through modeler, in chimera we can visualize the target through the residue name and also can label the residue by specific groups.

MOLECULAR DYNAMICS

According to the graph shown in the report, we find that the time taken for the root mean square deviation is 16 ps.

REFERENCES:
NCBI

www.ncbi.nlm.nih.gov/ SWISS PROT

www.expasy.org/group/swissprot PDB

www.rcsb.org/ MULTALIN

http://multalin.toulouse.inra.fr/multalin SAVS

http://nihserver.mbi.ucla.edu/SAVS/ PRODRG

http://davapc1.bioch.dundee.ac.uk/prodrg

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