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  1. 1. Faculty of Information ScienceTechnology LAB REPORT HBC 1019 - Biochemistry I 11-07-2012 ( Practical 8 ) Exploring Protein NAME : Osama Barayan ID : 1091105869
  2. 2. INTRODUCTION In this experiment we going to see whether animal from different species have similar protein structure. We used BLAST and CLUSTALW. We used BLAST to obtain sequences and the CLUSTALW to perform multiple sequence alignment. From this we can find how similar are their protein. METHOD 1. Obtaining sequences from BLAST. Triose phosphate isomerase is an enzyme that occurs in a central metabolic pathway called glycolysis. It is also known as an enzyme that demonstrates catalytic perfection. For this problem, you'll start with the sequence of triose phosphate isomerase from an organism and look for related proteins in the online databases. Here is the sequence of triose phosphate isomerase in FASTA format: > Triose phosphate isomerase (TIM) APSRKFFVGGNWKMNGRKKKNLGELITTLNAAKVPADTEVVCAPPTAYIDFARQKLDPKIAVAAQNCYKVTN GAFTGEISPGMIKDCGATWVVLGHSERRHVFGESDELIGQKVAHALSEGLGVIACIGEKLDEREAGITEKVVFE QTKVIADNVKDWSKVVLAYEPVWAIGTGKTATPQQAQEVHEKLRGWLKSNVSDAVAQSTRIIYGGSVTGATC KELASQPDVDGFLVGGASLKPEFVDIINAKQ a. Perform a protein-protein BLAST using the sequence above and answer the question below: I. What is the name of the organism that the enzyme belongs to? Triosephosphateisomerase II. What is the tissue originated from? Rabbit III. What is the accession number (from GenBank or etc) of this sequence? P00939.1 IV. State the authors and the title of the article journal involved by providing the details of the published journal and PubMed accession number. AUTHORS: Corran,P.H. and Waley,S.G. TITLE: The amino acid sequence of rabbit muscle triose phosphate isomerase JOURNAL: Biochem. J. 145 (2), 335-344 (1975) PUBMED: 1171682
  3. 3. b. Find a human homolog of this organism triose phosphate isomerase. isomerase,triosephosphate c. Does the NP_000356.1 entry represent a human ortholog of this organism triose phosphate isomerase? What is the percent identity between the two enzymes? Yes. 98% d. Use the human homolog from (b) to this organism’s enzyme. Click on the link on the left side of the record to bring up its GenBank entry. Select “FASTA” as the display format and click on the “Display” button. Copy the FASTA text and save it to a text file (if you are using a word processor, be sure to save the file in “text only” format). Save the text file (suggested name: TIM_FASTA.txt) for later use. e. Instead of trying to look through the entire BLAST output to find triose phosphate isomerase homologs from plants, bacteria, and archaea, you can use some options in BLAST to narrow your search. For example, look down the BLAST page for an option to select “Archaea” and then perform the BLAST search. Select one of the resulting sequences and save it in FASTA format. Repeat this process to get FASTA-formatted sequences for triose phosphate isomerases from a bacterial and plant (Viridiplantae) source. Combine the five FASTA- formatted sequences (rabbit, human, archaea, bacterial, and plant) in a single file (suggested name: TIM_5_FASTA.txt). This must be a simple text file with individual sequences separated by a blank line. f. Organize the five organisms in a table with the columns of species name, protein name, number of amino acids, % of identities, score and E-value. Species name Protien name No.ofa.a % of identities Score Evalue Rabbit Triosephosphateisomerise 248 99 504 1e-179 Human TriosephosphateIsomerase 250 98 498 0.0 T.aestivum triosephosphat-isomerase 253 64 326 2e-114 Krokinobacter triosephosphateisomerise 250 49 221 5e-72 euryarchaeote triosephosphateisomerise 249 38 160 1e-49 2. Multiple Sequence Alignment. A program called CLUSTALW will perform multiple sequence alignments on protein sets that are submitted in FASTA format. CLUSTALW is available as a command line program to be executed in a
  4. 4. UNIX environment (not very user-friendly). Fortunately, there are many Bioinformatics developers have integrate the program into graphical interface which facilitate the end-user to perform alignments. European Bioinformatics Institute is one of the examples that provide a web interface to perform CLUSTALW alignments: a. Go to the EBI site and submit your text file containing the five triose phosphate isomerase sequences in FASTA format on the input form page. There are many options for refining the alignment, but for now, use the default values. Be sure to enter your email address. The output of CLUSTALW can be accessed in many ways. The simplest version will be described here, but you are encouraged to explore other options (especially JaiView). In the simple text output, the sequences are optimally aligned and annotated: Residues that are identical in all chains are marked with an asterisk (*), those that are highly conserved are marked with a colon (:), and those that are semiconserved are marked with a period (.). From your multiple sequence alignment, how many identical residues did you find? Identify the residues, using the single-letter amino acid abbreviations. Classify these “identity” sites as polar, nonpolar, acidic, and basic amino acids. Do most of the “identities” fall into a single class of amino acids? RESULT rabbit --APSRKFFVGGNWKMNGRKKNLGELITTLNAAKVPAD--TEVVCAPPTAYIDFARQKL- 55 human GSAPSRKFFVGGNWKMNGRKQSLGELIGTLNAAKVPAD--TEVVCAPPTAYIDFARQKL- 57 T.aestivum---MGRKFFVGGNWKCNGTVEQVESIVNTLNAGQIASTDVVEVVVSPPYVFLPTVKGKL- 56 Krokinobacter----MRKNIVAGNWKMNNDLGATATLITALKQKKNEGN--AEVIIAPTFVNLYSAFDSLS 54 euryarchaeote---MSRTMLIAGNWKMNGLGADGGVLAKAVAVGSSAVG--CDLLICPPATLIYAVGEALS 55 *. ::.**** *. : :: . ::: .*. . : . * rabbit DPKIAVAAQNCYKVTNGAFTGEISPGMIKDCGATWVVLGHSERRHVFGESDELIGQKVAH 115 human DPKIAVAAQNCYKVTNGAFTGEISPGMIKDCGATWVVLGHSERRHVFGESDELIGQKVAH 117 T.aestivumRPEIQVAAQNCWVKKGGAFTGEVSAEMLVNLGVPWVILGHSERRSLMGESSEFVGEKVAY 116 KrokinobacterDSNITVAAQNMHQAESGAFTGEISAGMLKGIGVDTVILGHSERRAYFHESDELLAQKVDS 114 euryarchaeoteGSRVALGAQDCHAAESGAHTGDVSAEMLADAGCGFVIVGHSERRGEYGESDKIIHAKAQA 115 ..: :.**: .**.**::*. *: . * *::****** **.::: *. rabbit ALSEGLGVIACIGEKLDEREAGITEKVVFEQTK--VIADNVKDWSKVVLAYEPVWAIGTG 173 human ALAEGLGVIACIGEKLDEREAGITEKVVFEQTK--VIADNVKDWSKVVLAYEPVWAIGTG 175 T.aestivumALAQGLKVIACVGETLEQREAGSTMAVVAEQTK--AIADKIKDWTNVVVAYEPVWAIGTG 174 KrokinobacterALKNDMRIIFCFGEELEDRKAGKEENVVGSQLKNALFHLDASAWSNIVLAYEPVWAIGTG 174 euryarchaeoteVHRAGLIAVICVGETEAERDAGLAESVVAGQIG--GSLPQNGSAANSVIAYEPVWAIGTG 173 . .: : *.** :*.** ** * . :: *:*********** rabbit KTATPQQAQEVHEKLRGWLKSNVSDAVAQSTRIIYGGSVTGATCKELASQPDVDGFLVGG 233 human KTATPQQAQEVHEKLRGWLKSNVSDAVAQSTRIIYGGSVTGATCKELASQPDVDGFLVGG 235 T.aestivumKVASPAQAQEVHANLRDWLKTNVSPEVAESTRIIYGGSVTGASCKELAAQPDVDGFLVGG 234 KrokinobacterETASPEQAQDMHAFIRKTVAEKYNNEVAENVSILYGGSVKPANAKEIFEKPDVDGGLIGG 234 euryarchaeoteRTPTAEDVQIMHSHMRATVSASHGGDFGDQVRLLYGGSVKPGNSAELMGLTDVDGALVGG 233 ...:. :.* :* :* : . . ..:.. ::*****. ... *: .**** *:** rabbit ASLKP-EFVDIINAKQ---- 248 human ASLKP-EFVDIINAKQ---- 250
  5. 5. T.aestivumASLKP-EFIDIINAAAVKSA 253 KrokinobacterAALKADDFFDIVNAFA---- 250 euryarchaeoteASLVADDFLAIAMQCP---- 249 *:* . :*. * Number of identities: 68 POLAR NON-POLAR BASIC ACIDIC G=14 N=2 Q=3 T=2 S=4 H=2 C=1 Y=2 W=2 P=2 L=3 A=6 M=1 V=7 I=2 F=1 R=5 K=2 D=2 E=5 Polar=30 Non-polar=24 Basic=7 Acidic=7 Polar have the most identities with 30 identities, but non-polar only six identities less than polar. While both acidic and basic both have 7 identities. Phylogram
  6. 6. Cladogram The only different about this two tree is the meaning behind their branch length. The length of phylogram tree is according to the amount of character change but the length of cladogram tree mean nothing. From the tree we can see that the triose phosphate isomerase of these five species are link to each other. Human and rabbit is close to each other, while rabbit and euryarchaeote is far. DISCUSSION Although the protein is from the different animal, there is still some similarities. Some species might have higher identities with the other species compare to the other.contain different ratios of amino acids and higher concentrations of proteinsThis is base on how far are the species from each other in the evolution line.