PURIFICACION, MARYNOLD V.
CHEM 161.1 3L
2nd Semester AY 2012-1013
Donato, Lualhati M.
Diaz, Manuelle Marie C.
Date Submitted: March 8, 2013
Laboratory Instructor: Ms. Herra Grajo
Bioinformatics is the branch of biological science which deals with the study of methods for storing, retrieving and analyzing biological data, such as nucleic acid (DNA/RNA) and protein sequence, structure, function, pathways and genetic interactions. It is very important since it contains large amount of information regarding biomolecules that a human mind is not able to store and process such data. There are different data bases that can be used like National Center for Biotechnology Information (NCBI), European Molecular Biology Laboratory-European Bioinformatics Institute database (EMBL-EBI), GenBank (US-based), SwissProt/UniProt, DNA Data Bank of Japan (DDBJ), Entrez and PubMed. Basic Local Alignment Search Tool, or BLAST, is an algorithm for associating primary biological sequence information, like amino-acid sequences of various proteins or the nucleotides of DNA sequences. A BLAST search allows a researcher to compare a query sequence with a library or database of sequences, and identify library sequences that resemble the query sequence above a certain threshold. The BLAST program was designed by Stephen Altschul, Warren Gish, Webb Miller, Eugene Myers, and David J. Lipman at the NIH and was published in the Journal of Molecular Biology in 1990. On the other hand,ProtParam is a very useful softwarethat can compute various physico-chemical properties from a protein sequence. The parameters that can be computed by ProtParam include the molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index and grand average of hydropathicity (GRAVY).
At the end of this exercise, the student should be able to understand the concept and process of bioinformatics; to know the process on how to use computer programs related in biological information; and to apply these programs on different protein sequences and identify different informations using these programs.
The FASTA sequence of the given proteins namely; Myk, Gi, Glean, Astara, Niko, SR, Joma, Melai, Danne, Jay, Annie and Hani were analyzed using BLAST and ProtParam. BLAST showed the protein with that given sequence and its function was researched. ProtParam, on the other hand, showed the amino acid composition of the given protein, its theoretical IpH, estimated molecular weight and other pertinent information.
III. RESULTS AND DISCUSSION
Bioinformatics is the branch of biological science which deals with the study of methods for storing, retrieving and analyzing biological data, such as nucleic acid (DNA/RNA) and protein sequence, structure, function, pathways and genetic interactions. In this exercise, the computer program called Basic Local Alignment Search Tool (BLAST) was used to identify different protein sequences and determine the function of these proteins. Also, a computer program named ProtParam was used to determine the IpH and estimated molecular weight of the said proteins.
Different sequences of proteins were analyzed using these 2 algorithms to study their identities, properties and purposes. Table 1 show the list of the given protein sequences, their identity, their theoretical IpH and estimated molecular weight. The FASTA sequences of the different codes are also shown below.
Table 1. Identity, IpH and molecular weight of different protein sequences. Name| Identity| IpH| Molecular weight, g/mol|
Myk| NBD_sugar-kinase_HSAP70_actin superfamilyActin| 4.72| 10344.7| Gi| Pepsin A trypsin| 5.97| 7144.1|
Glean| AmyAC_family superfamilyAmylase A| 5.93| 10002.4| Astara| Protease| 8.97| 10595.0|
Niko| SERPIN superfamilySerpin ovalbumin| 6.24| 9899.4| SR| Glycosyltransferase_GTB_type superfamilyGlycogen phosphorylase| 4.65| 11336.7| Joma| Magainin| 5.21| 9931.1|
Melai| CASc superfamilyCaspase| 7.73| 12230.0|
Danne| Annexin superfamilyAnnexin| 6.14| 10022.5|
Jay| ADF_gelsolon superfamilyCofilin| 5.47| 11504.0|
Annie| Peptidase_C1ACathepsin| 5.80| 10982.2|
Hani| Euk_FerritinFerritin_like superfamilyFerritin| 5.06| 11519.9|
Actin forms microfilaments which are typically one of the most dynamic of the three subclasses of the eukaryotic cytoskeleton. In turn, this gives actin major functions in cells: * To form microfilaments to give mechanical support to cells, and provide trafficking routes through the cytoplasm to support signal transduction. * To allow cell motility in cells which undergo amoeboid motion using pseudopods and phagocytosis, for example of bacteria by macrophages. * In metazoan muscle cells, to be the scaffold on which myosin proteins generate force to support muscle contraction. In nonmuscle cells, to be a track for cargo transport myosins (nonconventional myosins) such as myosin V and VI. Nonconventional myosins use ATP hydrolysis to transport cargo, such as vesicles and organelles, in a directed fashion much faster than diffusion. Myosin V walks towards the barbed end of actin filaments, while myosin VI walks toward the pointed end. Most actin filaments are arranged with the barbed end toward the cellular membrane and the pointed end toward the cellular interior. This arrangement allows myosin V to be an effective motor for export of cargos, and myosin VI to be an effective motor for import.
Pepsin is an enzyme whose zymogen (pepsinogen) is released by the chief cells in the stomach and that degrades food proteins into peptides. The α-amylases (EC 18.104.22.168 ) (CAS# 9014-71-5) (alternative names: 1,4-α-D-glucan glucanohydrolase; glycogenase)are calcium metalloenzymes, completely unable to function in the absence of calcium. By acting at random locations along the starch chain, alpha-amylase breaks down ling-chain carbohydrates, ultimately yielding maltotriose and maltose from amyloase, glucose and “limit dextrin” from amylopectin. It can act anywhere on the substrate, α-amylase tends to be faster-acting than β-amylase. In animals, it is a major digestive enzyme, and its optimum pH is 6.7-7.0. In human physiology, both the salivary and pancreatic amylases are α-amylases.
A protease (also termed peptidase or proteinase) is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in thepolypeptide chain forming the protein. Serpins are a group of proteins with similar structures that were first identified as a set of proteins able to inhibit proteases. Glycogen phosphorylase catalyzes the rate-limiting step in glycogenolysis in animals by releasing glucose-1-phosphate from the terminal alpha-1,4-glycosidic bond.
Ovalbumin (OVA) is the main protein found in egg white, making up 60-65% of the total protein. Ovalbumin displays sequence and three-dimensional homology to the serpin superfamily, but unlike most serpins it is not a serine protease inhibitor. The function of ovalbumin is unknown, although it is presumed to be a storage protein. Ovalbumin is an important protein in several different areas of research, including:general studies of protein structure and propertiesbecause it is available in large quantities; studies of serpin structure and function since ovalbumin does not inhibit proteases which means that by comparing its structure with that of inhibitory serpins, the structural characteristics required for inhibition can be determined; in proteomics where it is used as a molecular weight marker for calibrating electrophoresis gel; and in immunology where it is commonly used to stimulate an allergic reaction in test subjects likean established model allergen for airway hyper-responsiveness, AHR.
Caspases, or cysteine-aspartic or cysteine-dependent aspartate-directed proteases are a family of cysteine proteases that play essential roles inapoptosis (programmed cell death), necrosis, and inflammation. Caspase 1/interleukin-1 converting enzyme is an enzyme that proteolytically cleaves other proteins, such as the precursor forms of the inflammatorycytokines interleukin 1-β and interleukin 18, into active mature peptides. It belongs to a family of cysteine proteases known as caspases that always cleave proteins following an aspartic acid residue. Caspase 1 has been shown to induce cell necrosis or pyroptosis and may function in various developmental stages. Studies of a similar protein in mouse suggest a role in the pathogenesis of Huntington's disease. Alternative splicing of the gene results in five transcript variants encoding distinct isoforms.
Annexins have been observed to play a role along the exocytotic pathway, specifically in the later stages, near or at the plasma membrane. Annexins have been found to be the later stages, near or at the plasma membrane. Annexins have been found to be involved in the transport and also sorting of endocytotic events. Annexin one is a substrate of the EGF (epidermal growth factor) tyrosine kinase which becomes phosphorylated on its N terminus when the receptor is internalized.
Cofilin is a ubiquitous actin-binding factor required for the reorganization of actin filaments. ADF/Cofilin family members bind G-actin monomers and depolymerize actin filaments through two mechanisms: severing and increasing the off-rate for actin monomers from the pointed end. "Older" ADP/ADP-Pi actin filaments free of tropomyosin and proper pH are required for cofilin to function effectively. In the presence of readily available ATP-G-actin cofilin speeds-up actin polymerization via its actin-severing activity (providing free barbed ends for further polymerization and nucleation by the Arp2/3 complex). As a long-lasting in vivo effect, cofilin recycles older ADP-F-actin, helping cell to maintain ATP-G-actin pool for sustained motility. pH, phosphorylation and phosphoinositides regulate cofilin’s binding and associating activity with actin The Arp2/3 complex and cofilin work together to reorganize the actin filaments in the cytoskeleton. Arp 2/3, an actin binding proteins complex, binds to the side of ATP-F-actin near the growing barbed end of the filament, causing nucleation of a new F-actin branch, while cofilin-driven depolymerization takes place after dissociating from the Arp2/3 complex. They also work together to reorganize microtubules in order to traffic more proteins by vesicle to continue the growth of filaments. Cofilin also binds with other proteins such as myosin, tropomyosin, α-actinin, gelsolin and scruin. These proteins compete with cofilin for actin binding. Сofilin also play role in innate immune response. Cathepsins have a vital role in mammalian cellular turnover, e.g. bone resorption. They degrade polypeptides and are distinguished by their substrate specificites. Ferritin serves to store iron in a non-toxic form, to deposit it in a safe form, and to transport it to areas where it is required. Knowing the protein sequence gives many advantages in studies especially dealing with medicine. The protein of interest whether it is the cause of the abnormality or the cure for abnormality can be identified with just few clicks.
The reasons behind similarity of protein sequences despite diversity of source organism is because even though all protein families have distinct functional compositions across different species, some conserved functional features among family members included a shared reaction mechanism, cofactor usage, and/or ligand specificity.
IV. SUMMARY AND CONCLUSION
Bioinformatics is the branch of biological science which deals with the study of methods for storing, retrieving and analyzing biological data, such as nucleic acid (DNA/RNA) and protein sequence, structure, function, pathways and genetic interactions. It is very important since it contains large amount of information regarding biomolecules that a human mind is not able to store and process such data. Basic Local Alignment Search Tool (BLAST), an algorithm for associating primary biological sequence information, like amino-acid sequences of various proteins or the nucleotides of DNA sequences; and ProtParam, a very useful software that can compute various physico-chemical properties from a protein sequence. Such parameters include the molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index,