BOC334: Proteomics
Practical 2
Surname……………………………. Initials:………… Student nr..........................
TYPE YOUR ANSWERS IN THE BLOCKS
You can delete the instructions to save space
Identifying the product of an unknown gene and finding its relatives

Open EXPASY web site by typing http://www.expasy.org

Click “proteomics”

Select “translate” under “Tools”
Exercise 1
[6]
>exercise_1
ctgattagcctgagcggcgcggaagcggatctgctggcgaaaagctgggcgccggtgtttgcgaacaaagatgcgaa
cggcgataactttctgattgcgctgtttgaagcgtttccggatagcgcgaacttttttggcgattttaaaggcaaaa
gcattgcggatattcgcgcgagcccgaaactgcgcagcgtgagcagccgcattgtgaaccgcctgaacgattttgtg
ggcaacgcggcggatgcgggcaaaatggcgggcatgctggatcagtttagcaaagaacatgtgggctttggcgtggg
cagccagcagtttgaaaacgtgcgcagcatgtttccgggctttgtgagcagcgtggcggcgccgccggcgggcgcgg
atgcggcgtggggcaaactgtttggcctgattattgatgcgctgaaaaaagcgggcaaa
Copy and paste the >exercise_1 DNA sequence only into the sequence box, select
“verbose output” followed by “translate”.
Question 1: Can you identify possible reading frames that will code for proteins?
Copy and paste the results output here (change the Met to M) of the identified ORFs.
Highlight the possible ORFs.
Go to NCBI home page (http://www.ncbi.nlm.nih.gov/) and select BLAST followed by
BLASTp. Individually paste your putative protein sequence/s into the sequence box.
Run BLASTp using the default parameters against the nr database.
Question 2: Which ORF codes for a functional protein? Motivate (tip: look at the
e-values).
Type the answer here
Question 3: What protein is this? Motivate.
Type the answer here

Download six sequences of your functional protein and paste them into a file.
Prepare a FASTA format file in Word or Notepad. Use the first hit, the last one
(that is the same as your query sequence) and other hits in between with a range
of different e-values.

Go to http://www.ebi.ac.uk/Tools/msa/clustalw2/
Exercise 2
[5]
Paste all the sequences in FASTA format into the sequence box. Run ClustalW using
default parameters.
Question 4: Can you identify conserved amino acids?
Paste the results of the ClustalW here and highlight the conserved amino acids. Use
font size 9 when pasting into Word document.
Question 5: One of the residues is a histidine. Find out in the literature why this
is important for the function of the myoglobin?
Type your answer here
Finding related proteins for which structures are known
Repeat the BLAST search as before using the sequence of your functional protein but
use the PDB database.
Exercise 3
[5]
On the BLAST output screen, click on any of the links on the left hand of the screen
next to one of the results.
Question 6: Copy and paste the display on the screen.
Copy and paste here
Highlight the name of the molecule and the species from which it originated
Go to http://www.pdb.org
Type the letters corresponding to the code for the protein in the search window e.g.
1uc3
Download the pdb file.
Open Rasmol and load the pdb file.
Question 7: Prepare a picture using Rasmol which illustrates the most important
features of the molecule which you previously saw in practical 1.
Copy and paste (do “print screen”) the picture on the screen and point out the
most important features.
Copy and paste here
Highlight the name of the molecule and the species from which it originated
Exercise 4
[4]
ClustalW Alignment:
 Select any three sequences (from previous search) and download them into a
new file as before in FASTA format.

Align these three sequences with your original query sequence using ClustalW

Cut and paste the relevant alignments into this document as before.
Paste results here
Protparam:

Go
back
to
the
proteomics
and
sequence
analysis
tools
(http://www.expasy.org/tools/)

And find the www.expasy.org/tools/protparam.html page.

Individually paste two sequences into the query window and run the tool. Use
the original query sequence and any other sequence you obtained from your
searches.

Cut and paste your results into this file and save the file.
Paste results here