Faculty of Information Science & Technology

LAB REPORT

HBC 1019 - Biochemistry I

11-07-2012

( Practical 8 )

Exploring Protein

NAME : Osama Barayan

ID : 1091105869

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)

APSRKFFVGGNWKMNGRKKKNLGELITTLNAAKVPADTEVVCAPPTAYIDFARQKLDPKIAVAAQNCYKVTNGAFTGEISPGMIKDCGATWVVLGHSERRHVFGESDELIGQKVAHALSEGLGVIACIGEKLDEREAGITEKVVFEQTKVIADNVKDWSKVVLAYEPVWAIGTGKTATPQQAQEVHEKLRGWLKSNVSDAVAQSTRIIYGGSVTGATCKELASQPDVDGFLVGGASLKPEFVDIINAKQ

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? Triosephosphate isomerase

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

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.of a.a % of identities

Score Evalue

Rabbit Triosephosphate isomerise

248 99 504 1e-179

Human Triosephosphate Isomerase

250 98 498 0.0

T.aestivum triosephosphat-isomerase

253 64 326 2e-114

Krokinobacter triosephosphate isomerise

250 49 221 5e-72

euryarchaeote triosephosphate isomerise

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 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: http://www.ebi.ac.uk/clustalw/.

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?

RESULTrabbit --APSRKFFVGGNWKMNGRKKNLGELITTLNAAKVPAD--TEVVCAPPTAYIDFARQKL- 55human GSAPSRKFFVGGNWKMNGRKQSLGELIGTLNAAKVPAD--TEVVCAPPTAYIDFARQKL- 57T.aestivum ---MGRKFFVGGNWKCNGTVEQVESIVNTLNAGQIASTDVVEVVVSPPYVFLPTVKGKL- 56Krokinobacter ----MRKNIVAGNWKMNNDLGATATLITALKQKKNEGN--AEVIIAPTFVNLYSAFDSLS 54euryarchaeote ---MSRTMLIAGNWKMNGLGADGGVLAKAVAVGSSAVG--CDLLICPPATLIYAVGEALS 55 *. ::.**** *. : :: . ::: .*. . : . *

rabbit DPKIAVAAQNCYKVTNGAFTGEISPGMIKDCGATWVVLGHSERRHVFGESDELIGQKVAH 115human DPKIAVAAQNCYKVTNGAFTGEISPGMIKDCGATWVVLGHSERRHVFGESDELIGQKVAH 117T.aestivum RPEIQVAAQNCWVKKGGAFTGEVSAEMLVNLGVPWVILGHSERRSLMGESSEFVGEKVAY 116Krokinobacter DSNITVAAQNMHQAESGAFTGEISAGMLKGIGVDTVILGHSERRAYFHESDELLAQKVDS 114euryarchaeote GSRVALGAQDCHAAESGAHTGDVSAEMLADAGCGFVIVGHSERRGEYGESDKIIHAKAQA 115 ..: :.**: .**.**::*. *: . * *::****** **.::: *.

rabbit ALSEGLGVIACIGEKLDEREAGITEKVVFEQTK--VIADNVKDWSKVVLAYEPVWAIGTG 173human ALAEGLGVIACIGEKLDEREAGITEKVVFEQTK--VIADNVKDWSKVVLAYEPVWAIGTG 175T.aestivum ALAQGLKVIACVGETLEQREAGSTMAVVAEQTK--AIADKIKDWTNVVVAYEPVWAIGTG 174Krokinobacter ALKNDMRIIFCFGEELEDRKAGKEENVVGSQLKNALFHLDASAWSNIVLAYEPVWAIGTG 174euryarchaeote VHRAGLIAVICVGETEAERDAGLAESVVAGQIG--GSLPQNGSAANSVIAYEPVWAIGTG 173 . .: : *.** :*.** ** * . :: *:***********

rabbit KTATPQQAQEVHEKLRGWLKSNVSDAVAQSTRIIYGGSVTGATCKELASQPDVDGFLVGG 233human KTATPQQAQEVHEKLRGWLKSNVSDAVAQSTRIIYGGSVTGATCKELASQPDVDGFLVGG 235

T.aestivum KVASPAQAQEVHANLRDWLKTNVSPEVAESTRIIYGGSVTGASCKELAAQPDVDGFLVGG 234Krokinobacter ETASPEQAQDMHAFIRKTVAEKYNNEVAENVSILYGGSVKPANAKEIFEKPDVDGGLIGG 234euryarchaeote RTPTAEDVQIMHSHMRATVSASHGGDFGDQVRLLYGGSVKPGNSAELMGLTDVDGALVGG 233 ...:. :.* :* :* : . . ..:.. ::*****. ... *: .**** *:**

rabbit ASLKP-EFVDIINAKQ---- 248human ASLKP-EFVDIINAKQ---- 250T.aestivum ASLKP-EFIDIINAAAVKSA 253Krokinobacter AALKADDFFDIVNAFA---- 250euryarchaeote ASLVADDFLAIAMQCP---- 249 *:* . :*. *

Number of identities: 68

POLAR NON-POLAR BASIC ACIDICG=14N=2Q=3T=2S=4H=2C=1Y=2

W=2P=2L=3A=6M=1V=7I=2F=1

R=5K=2

D=2E=5

Polar=30 Non-polar=24 Basic=7 Acidic=7Polar 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

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 proteins This is base on how far are the species from each other in the evolution line.