1. Faculty of Information ScienceTechnology
LAB REPORT
HBC 1019 - Biochemistry I
11-07-2012
( Practical 8 )
Exploring Protein
NAME : Osama Barayan
ID : 1091105869

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. 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. 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?
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. 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. 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.