> Contents Introduction of Protein Types of Protein Methods of Protein Structure Prediction ~ Threading ~ Homology Modelling Rosetta Application and Lit RNA: IF you cant hep moke profens, References get out of the kitchen, onvoarze ‘COMPUTATIONAL PREDICTION OF PROTEIN ‘STRUGTURE> Introduction * Protein : A polymeric chain of amino acid residues constitutes proteins, * Protein structure : Protein sequence folds into a unique shape that minimizes its free potential energy. + A protein's structure is primarily made up of long chains of amino acids + The arrangement and placement of amino acids give proteins certain characteristics, * Poly, ide chains are synthesized by linking together amino a * A protein is created when one or more of these chains fold in a specific way. oxvoarze ‘COMPUTATIONAL PREDICTION OF PROTEIN 3 ‘STRUGTURE> Types of Protein + The structure of the protein is classified at 4 levels O Protein Structure ad CT Gad acl = Balad Gi-helices f= sheets aawoarze ‘COMPUTATIONAL PREDICTION OF PROTEIN. ‘STRUGTURE> Methods of Protein Structure Prediction + There are mainly two methods : 1) Computational methods 2) Experimental methods Methods Computational Methods Experimental Methods + Threading Method * Homology Modeling Method * X-ray Crystallography + NMR Spectroscopy + Cryo-EM avoarze ‘COMPUTATIONAL PREDICTION OF PROTEIN ‘STRUCTURE OA) Threading Method + Also called as Fold Recognition Method. * Searched for a structural fold database to find the best matching structural fold using energy based criteria, + Ttuses dynamic programming and heuristic approaches to calculate energy of raw model. + Lowest energy fold that correspond to the structurally a group of most compatible fold. hk a re => f acioarze ‘COMPUTATIONAL PREDICTION OF PROTEIN 6 ‘STRUCTURE.Conti..... he onvoarze “* Steps involved in threading method Coens 2 The design of scoring function Cnet | ‘COMPUTATIONAL PREDICTION OF PROTEIN ‘STRUGTUREConti..... 1) Construction of structure template database * Select the protein structures from a protein structure database. * When selecting the protein structure, we need to make sure that we avoid protein structures which have high sequence similarity, * PDB, FSSP, SCOP and CATH 2) The design of the scoring function * Mutation potential, pairwise potential, secondary structure compatibi s and gap penalties are the main things a scoring function should consist of, + Ascoring function should measure the fitness between the template and the target sequence. ‘COMPUTATIONAL PREDICTION OF PROTEIN 8 ‘STRUGTURE.Conti..... 3) Threading alignment + The scoring function has to be optimized so that the target sequence can be aligned with each of the structure templates + In threading- based structure prediction program, both sequence are refined to account for potential errors and inaccuracies. 4) Threading prediction * Choose a threading alignment that is statistically likely as the threading protein, + Structural model is constructed by using the selected structural template and placing the backbone atoms of the target sequence on the structural templates aligned back one positions. agioarze ‘COMPUTATIONAL PREDICTION OF PROTEIN 9 ‘STRUCTUREConti..... Advantages * Itis moderately successful. + Itis good for proteins with less than 100 residues, Disadvantages * This methodology assumes that the current structural library consists of all the possible confirmation that could possibly be deciphered experimentally. * High computational cost is involved to screen a Library of thousands of possible folds. + Energy functions are simplified for efficient calculations and results are therefore compromised. ‘Threading softwares : Modeller, Hhpred, RaptorX, Phyre and Phyre2, FALCON, MUSTER ‘COMPUTATIONAL PREDICTION OF PROTEIN 10 ‘STRUGTUREConti... B) Homology Modeling Method + Also called comparative modeling. * Predict protein structure based on sequence homology with known structure. + If two proteins share a high enough sequence similarity they are likely to have very similar three dimensional structure. * Modeling server :- modbase, Swiss- model, etc, aawoarze ‘COMPUTATIONAL PREDICTION OF PROTEIN " ‘STRUGTUREConti Steps involved in Homology Modeling method Template recognition and Initial alignment Alignment correction. Loop modeling Side chain modelling Model optimization Model validation avoarze ‘COMPUTATIONAL PREDICTION OF PROTEIN ‘STRUCTUREConti..... 1) Template Recognition and Initial Alignment * Searching of the PDB for homologous protein which consists of determined structures. + BLAST Seareh (Basic Local Alignment Search Tool) Ivis a search algorithm which can compare our query sequence with the sequence present in a Library or database. And it gives us the sequence which match our query sequence. It can search amino acid sequence of protein, nucleotides of DNA and RNA sequences. + FASTA Search(Fast-A) It is a software which is primarily used for DNA and protein sequence alignment. Ithas.a high speed of execution, First, it marks the potential matches using the pattern of word hits and word-to-word matches of a given length, It is also famous for its FASTA format which has become widely used in bioinformatics domain. acioarze ‘COMPUTATIONAL PREDICTION OF PROTEIN 8 ‘STRUCTURE,Conti. 2) Alignment Correction * The alignment of two sequences of two proteins can be difficult, where the percentage sequence identity is low. * One can then use the other sequences present from the homologous protein to find a solution. + Ex - It is nearly impossible for the sequence LTLTLTLT to be aligned with sequence YAYAYAYAYA. So we find a third sequence TYTYTYTYT which can be easily aligned with both of them, 3) Loop Modeling + After the sequence alignment step, duc to insertion and deletion there are often regions created that lead to gaps in the alignment. Loop modeling is used to fill in these gaps, but it is comparatively less accurate ‘COMPUTATIONAL PREDICTION OF PROTEIN “ ‘STRUCTUREConti... + The bwo main techniques are used to approach the problem a) The database searching method : It involves going through the database of known protein structures and finding loops and then superimposing these loops onto the two stem region of the target regions of the target protein, b) The ab initio method ; In this method, various loops are generated randomly. And then, it searches of a loop in those loops which has reasonably low energy. + ModLoop is.a web server for automated modeling of loops in protein structures, 4) Model Optimization * Itis used to evaluate the protein-ligand interactions at the active sites and also the protein-protein interactions at the contact interface, aurea ‘COMPUTATIONAL PREDICTION OF PROTEIN. % ‘STRUGTURE.Conti..... By searching every possible combination for every torsion angle of the side chain, we can select the one which has the lowest interaction energy with neighboring atoms. * Model optimization is done so that the overall conformation of the molecule has the lowest possible energy potential. * This is done by adjusting the relative position of the atoms which leads to a better chance of finding the true structure. Energy = Stretching Energy + Bending Energy + Torsion Energy + Non-Bonded Interaction Energy ‘COMPUTATIONAL PREDICTION OF PROTEIN acioarze ‘STRUGTURE.Conti..... 6) Model Validation Every homology model contains errors for which there are two main reasons. The first error occurs due to sequence identity, if it is more than 90% or less than 30% of the template and target protein, If the sequence identity is more than 90%, than it can be compared with the structures which are determined through crystallography. Else if it is less than 30%, then major errors can occur. The second major reason for errors is the errors present in the templates itself. Advantages It can find the location of alpha carbons of key residues inside the folded protein. It can hypothesize strueture-functions relationship. ‘COMPUTATIONAL PREDICTION OF PROTEIN " ‘STRUCTUREConti 3. 4 Mutagenesis experiments can be guided using this. The putative active sites, binding pockets and ligands can be identified using the position of the conserved regions of the protein surface. Disadvantages ‘The side-chain positions and predicting conformations or deletions are not possible with homology models. Homology models are only useful for drug designing and development process only if the sequence identity of the target protein is more than 70% with the template. Because template with less than 70% identity cannot be used in modeling and ligand docking studies, ‘STRUCTUREConti..... * Error Sources In Homology Modelling Incorrect sequence alignment is among the most devastating error in homology modelling. Incorrect choice of template may happen, especially for multidomain proteins. The loop regions can be built incorrectly. ‘The person doing the modeling can also make some errors, but this type of errors can be hard to predict and it can include any kind of errors. There can also be errors present in the template itself. Homology Modelling Softwares : Int\FOLD, RaptorX, Biskit, MODELLER, SWISS- MODEL. acioarze ‘COMPUTATIONAL PREDICTION OF PROTEIN. 19 ‘STRUCTURE,> Rosetta It is a Web server for protein 3d structure prediction. Rosetta development began in the laboratory of Dr. David Baker at the University of ion tool. Washington as a structure predi Mini threading method Breaks down the query sequence into many short segments (3 to 9) Predicts the secondary structure of small segments using HMM Segments with assigned secondary structure are subsequently assemble into a 3D configuration. Random combination of fragments, a large number of models are built and their over all energy potential calculated. ‘COMPUTATIONAL PREDICTION OF PROTEIN 20 ‘STRUCTURE> Application and Limitation of Methods for structure prediction These methods provide structural details to different extent, * Homology Modeling can provide atomic level details of target protein, threading can help only to judge the fold of the protein, High and medium level homology models with sequence identity > 30% are useful in refining functional prediction such as ligand binding. + Folds predicted by threading could be used in supporting site-directed mutagenesis experiments, designing of stable crystallizable variants and in refining NMR structures. * In predicting functional relationships from structural similarity and for identification of patches of conserved surface residues. avoarza ‘COMPUTATIONAL PREDICTION OF PROTEIN 2 ‘STRUCTURE,>» References 2. Jisna, V. & Jayaraj, P. B. (2021), Protein Structure Predietion: Conventional and Deep Learning Perspectives. The protein journal, 40(4), 522-544. htips://doi.org/10.1007/s10930- 021-10003-y https://www.slideshare.net/ashharnomani/computational-predi ion-of-protein| pptx#27 ‘COMPUTATIONAL PREDICTION OF PROTEIN ‘STRUGTURETHANK o YOU i