Bioinformatics and Molecular Modelling - Research Paper Example

Bioinformatics and Molecular Modelling Bioinformatics and Molecular Modelling Part I and II Introduction Lipases are glycerol estersthat often act on the triacylglycerols so that they cab release glycerol and acids. The major part of the lipases are used in industrial processes today are stemmed from animal or microbial sources. Notably, plant enzymes have some advantages over animals and microbial enzymes since they are available from natural sources with low costs and ease of purification. They widely exist in plant and microbial species, but with poor characterization of structure. Plant lipases are often considered to be involved in regulating certain plant growths and developments (Bos and Laxminarayan, 2011; 42). They are mainly found in seeds where triglycerides are stored in the form of intracellular structures or the oil bodies. Lipases usually hydrolyze triglycerides to fatty acids and glycerol that produces energy needed for seed germination. The plant lapses are usually classified into three main groups with the first group consisting of the triacylglycerol hydrolases that are mainly found in seeds. Their study is vital since they are responsible for seed alteration especially during storage. The second group is the acylhydrolases that are found in various plant tissues. They often exhibit limited specificity for their substrates; therefore, they are unable to hydrolyze triglycerides. However, they are cable to catalyze some esterification processes or reactions (Appel and Feytmans, 2009; p. 68). The profound acylhydrolases include phospholipases A and B, sulfolipases, glycolipases, and monoglyceride lipases. The last group in this category is the phosphorlipases that involve plant metabolism, degradation, and rearrangement. Other than the above classification, the recent studies have led to different classification of lipases based on comparison of the sequences of their amino acid among other fundamental biological and physicochemical properties (Gupta, 2007; 34). This modern classification led to eleven subfamilies. Despite being a member of many protein families, the lipases often have similar architecture that is described by the β-hydrolase fold. The activities of all lipases often rely on the catalytic triad that is usually fromed by the Asp, Ser, and His residues. In the sequence of the amino acid especially involving α/β hydrolases, these three residues often follow the user-Asp-His order. Additionally, lipases often share the consensus sequence defined by the Gly-Xaa-Ser-Xaa-Gly where X may be a residue of an amino acid (Bos and Laxminarayan, 2011; p. 33). The three dimensional structure of any protein molecule often provides valuable insight into the molecular function, organization, docking stimulation, and the effective designing of drug experiments. The lack of an experimentally determined crystal structure, the homology modeling may be used to provide an opportunity in obtaining a reasonable 3D model. Currently, the 3D models often provide a perfect means of predicting the structure of biomolecules since it yields models that are suitable for a wide application spectrum that are structurally based thereby providing molecular design for mechanism investigation. The 3D approach is capable of providing a reasonable structure model that is often related to template that shares more than 25 percent sequence identity. An Arabidopsis thaliana lipase model is greatly proposed to investigate the model organism mainly in the plant biology since it is relatively small and and it is genetically tractable genome. Methods The Target and Template Proteins The model is perfect in determining adequate template for the homology modeling for the Arabidopsis thaliana. This sequence allows the alignment of amino acid sequence against the protein data bank (PDB) and this is performed by means of BLAST algorithm. According to the sequence algorithm, both the template (1HLG) and the target share 31 percent of the sequence identity. In this case, the protein structures are highly conserved compared to DNA structures (Crichton and Boelaert, 2001; 141). In this method, the similarities of the detectable sequence levels are often significant in the structural similarities. As per the significant e-value and alignment of the investigated template, the human lipases can be put under investigation or study (PDB ID: 1HLG) (Appel and Feytmans, 2009; p. 58). In this case, the target protein amino acid is to be extracted from the LIPABASE database. Notably, this database provides physicochemical, taxonomic, and molecular information about the “true” lipase from the different species. The template used in the practical is the human gastric lipase marked or coded as (PDB ID: 1HLG). This template has 461 amino inclusive of other two chains: A and B leading dimmer. The method will allow resolution and the R-value of the template will be selected from the PDB at 3OA and 0.210 respectively. Homology Modeling This model was contracted using the Swiss-PDB Viewer version 4.0.1. This application often provides the user with a friendly interface thereby allowing analyses of several proteins at once or at the same time. Moreover, the model allows identification of one or several unknown protein structures that are likely to resemble the query sequence structure. It also produces an alignment that maps residue on the query sequence to the residue in the template sequence. Different types of modeling requests can be made the Swiss-PDB program including the alignment mode, project mode, and automated mode. The alignment mode often allows the user to test many alternative alignments and evaluate their quality towards attaining optimal results (GU and Bourne, 2011; p. 40). However, it is often a difficult model in situations where there cannot be a correct alignment between the target and the template. Nonetheless, manual manipulation, visual inspections, and adoption of sequence based methods can improve the quality of the result significantly. The project mode is essential in determining the Arabidopsis thaliana lipase modeled structure. Notably, the project files often contain the structure of superposed template and the alignment between the target and template. This file is usually generated in the program Deep View through the use of workspace template selection tools. Model Validation It is quite vital to use accurate homology model; thus, it is often profound to build appropriate steps in the process to ensure high quality model is used in the practice. The accuracy is the model is often subjected to a series of tests towards verification. Ramachandran plots allow stereochemical quality evaluation through the RAMPAGE server and amino acid environment. This is used to access the template using the 3D and Errat. Docking The natural substrate lipase is triacylglyceride to the validate the active site or the architecture of the Arabidopsis thaliana lipase model to allow examination of its possible modes of interaction with other ligands. In the homology model, the triacylglyceride substrate is docked using the HEX v.5.1 docking environment and this may be as well be set as the default parameters. The Hex is a macromolecular docking tool and it can as well superpose pairs of molecules in 3D shapes. This property also helps in determining or tidying the ligand-protein interactions. This process is often suitable in assuring perfect results especially in predicting substrate binding sites (Gromiha, 2010; p. 82). Notably, the post processing and correlation output for ligands and receptors are kept based on shape, minimization of molecular mechanics, and electrostatic potential (Bos and Laxminarayan, 2011; 92). Full rotation docking is often done to allow full flexibility for the ligands while maintaining the position of the receptors in a fixed space. Docking parameters that are involved in the practice include steric scan, Fourier transformation, refinement of the complex, and finally search for ligand binding sites. Results and Discussion Figure 1. Ribbon presentation of a 3D structure of Arabidopsis thaliana lipase enzyme in Rasmol Homology Models and Validation According to the study, the sequence identity is greater than 25 percent between proteins thereby indicating similar 3D structures. In this case, the template and the target proteins share 32 percent of the sequence identity (Acton, 2012; p. 185). Moreover, the alignment of the Arabidopsis thaliana lipase model was a creation of the human gastric lipase (PDB: 1HLG) for the template of the model leading Fig. 1. Therefore, the modeled enzyme is a monomer that is folded in the domain of α/β that has eight stranded β sheet that are flanked with 22 α helices. Ψ = 0 General/Pre-Pro/Proline favored General/Pre-Pro/Proline allowed Glycine favored Glycine allowed Figure 2. Ramachandran plot indicating residues in favored, allowed, and outlier regions The above figure (Fig. 2) Shows the regions of possible and formation by c (psi) and w (phi) angles. This is a conventional term representing the torsion angle on either side alpha carbon of the peptide. The 91.5 percent division of the plot allows six percent allowed and 2.5 percent outliers. Therefore, the result herein is significant since the favored residues are within a region of less than 90 percent. Moreover, this is an indication that the model is built on a good quality (Appel and Feytmans, 2009; p. 68). The use of the Errat plot enables the assessment of the arrangement of different types of atoms that are in line with each other in the protein models (as indicated in Fig. 3). The Errat technique is quite sensitive; hence, it often allows identification of incorrectly folded regions within the preliminary protein models. The ERRAT is the overall quality factor that indicates the non-bonded atom interactions; thus, higher usually indicates high quality (Acton, 2012; p. 345). Normally, the accepted high quality values often range in the region less the 50; however, in our case it is 58.989. Error values 99% 95% 0 35 75 95 125 145 165 185 205 225 245 265 Residue (window center) Figure 3. The plot for the Arabidopsis thaliana lipase model. The black bars indicate the misfolded region that is located from the active sites while the gray bars indicate error regions that fall between 95% and 99% and the white bars represent the regions of less error rates for the protein foldings This program enables the analysis of the compatibility of atomic models in a three dimensional (3D) of any given amino acid sequence (ID). The scores of the analysis often ranges from -1 (which is a bad score) to +1 (best score). The verification diagram for the score for the lipase model is as in the figure 4 below 6 4 Verify scores 2 0 -2 1 30 60 90 120 150 180 210 240 270 300 Amino Acid Index Figure 4. The verification diagram for validating the Arabidopsis thaliana lipase model. The homology model for the Arabidopsis thaliana lipase model was validated for stereochemical as well as the amino acid environment quality (Acton, 2012; p. 245). The proper validation was achieved through docking using the natural substrate that aimed at determining the key residues in the ligand binding. Human and mouse The chicken’s CALCULATIONS Calculate Folding Energies of Human, Rat and Mouse TfR IRE From the basic understanding of the RNA folding program, the CAGUGC loop sequence of TfR IRE is singlely unpaired and forms a bulge position with a predicted free energy of folding of ΔG = −5.9 kcal/mol. Looking the rats sequence there are 620 nt loops that translates to (-5.9*620) kcal/mol = -3658.00 kcal/mol. If the same basis is tramslated to human that 250nt the energy will be (-5.9*250)= -1475 kcal/mol. Mouse’s energy (-5.9*580)= -3422 kcal/mol. Comparison between the Sequences of the Second and Third IREs Iron often regulates the level of the mRNA encoding the transfering receptor (TfR) and the regulation is intern regulated by a portion of the 3’ untranslated region (UTR) in the TfR transcript. The human TfR mRNA part of 3’ UTR contain five elements with structural similarities to the iron responsive elements (IRE) that are usually found as single copies in the 5’ URT of the mRNA. Therefore, both the first and second IREs contain similar elements (Abraham, 1999; p. 452). Notably, the same elements are countained in the chicken’s TfR mRNA in the 3’ UTR that forms the first IRE. The cytosolic extract from human cell lines are found in the RNase T1 protection that contain the IRE binding protein that is capable of specific interactions with the TfR 3’ UTR from human. The removal of the protecting protein that protects RNA can easily be digested with RNase T1 thereby yielding oligoribonucleotide fragments that are characteristic of the two IREs countained in the TfR 3’ UTR. Finally, it worth noting that the two IRE often interacts where IRE-binding proteins often iteract with the ferritin IRE. References Abraham, N. G. (1999). Molecular biology of hematopoiesis 6: [proceedings of the Eleventh Symposium on the Molecular Biology of Hematopoiesis, held June 25-29, 1998, in Bormio, Italy. New York [u.a.], Kluwer Academic/Plenum Publishers. Acton, Q. A. (2012). Issues in bioengineering and bioinformatics. Atlanta, Ga, ScholarlyEditions. Appel, R. D., & Feytmans, E. (2009). Bioinformatics: a Swiss perspective. Singapore, World Scientific Pub. Bos, L., & Laxminarayan, S. (2011). Future visions on biomedicine and bioinformatics a liber amicorum in memory of Swamy Laxminarayan 2. Heidelberg, Springer. Crichton, R. R., & Boelaert, J. R. (2001). Inorganic biochemistry of iron metabolism: from molecular mechanisms to clinical consequences. Chichester, Wiley. Gromiha, M. M. (2010). Protein bioinformatics from sequence to function. Amsterdam, Academic Press/Elsevier. Gu, J., & Bourne, P. E. (2011). Structural Bioinformatics. Hoboken, John Wiley & Sons. Gupta, P. K. (2007). Genetics: lassical to modern. Meerut, India, Rastogi Publications. Read More