BOC334: Proteomics
Practical 3
Total: 20
Surname:……………………………. Initials:………… Student nr:..........................
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In this exercises, you have an unknown protein sequence and be required to use
bioinformatics tools available on the internet to assign some functions to the protein. In some
cases it will be possible but in others you may have to speculate. It is important that you use
the available information to formulate your answers. Motivate your answers properly.
Godfrey, a PhD student, is studying enzyme systems responsible for triacylglycerol
hydrolysis in a thermophilc organism, Geobacillus thermoleovorans (thermoleovorans
means “hot oil eater”), which was isolated from a deep SA gold mine at a depth of about
3.2 km underground. The current hypothesis is that the organisms secrets a “scout”
enzyme which will hydrolyse any TAG in the environment after which the products of
the hydrolysis induce large amounts of enzyme. A possible candidate enzyme in a
similar organism was purified and the amino acid sequence was determined by a group
in Korea. Godfrey reverse translated the amino acid sequence and designed PCR
primers to amplify the corresponding gene in G. thermoleovorans. Degenerate primers
produced an amplicon of the correct size. After cloning and sequencing, the nucleotide
sequence suggested the following amino acid sequence, >GEOTM1. In this exercise you
will try to do a functional annotation of >GEOTM1 using different bioinformatic
techniques.
1. Use the amino acid sequence >geotm for the following exercises:
>GEOTM
MLKRIIKNERGLTLIELLAVIVILGIVAAIAIPAIGGLINKSKDDAKIAEGIQIINAAKLYMTANTPANFPHTLSKTELDSYLDNVKDQDYEVEVSKDGVNGKYSYVLKK
HDANTVLNDAELTEEELQNKKSDK
2. Go to the blast website at NCBI (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi)
3. Select blastp and copy and paste the sequence (ONLY!) into the query window.
4. Select the nr database
Question 1: What function can you assign to the protein?
(1)
Type your answer here
Question 2: Motivate your answer.
Type your answer here
(1)
5. Go to the Expasy website (http://ca.expasy.org/tools/)
6. Select “Proteomics” on the left panel. Under “Tools” select ProtParam
7. Copy and paste the sequence only into the query window and click on the compute
parameters button.
Question 3: What is the molecular weight and pI?
Type your answer here.
(2)
8. Paste the following link into your browser: http://www.cbs.dtu.dk/services/SignalP/
9. Paste your sequence into the query box and run the algorithm for Gram positive organisms.
Question 4: Is the protein secreted or not? Motivate your answer by pasting the graph
and scores in the box. Explain the information and your answer. HINT: click on the
“explain the output” link at the bottom of the page.
(4)
Type your answer here
10. On the ExPASy Bioinformatics Resource Portal, select PROSITE under “Databases”. On
the PROSITE site, select ScanProsite. Paste the sequence (only) into the box (sequences to
be scanned) and click on “start the scan”.
Question 5: What functional motif did you detect? Follow the link just below the hits by
patterns: [1 hit (by 1 pattern) on 1 sequence] line on the screen. Paste the amino acid
sequence of the motif in this box.
(2)
Type your answer here
11. On the ExPASy Bioinformatics Resource Portal, select “ProtScale” under Tools.
12. Paste the sequence into the query window and run a hydrophobicity plot using the
algorithm of Kyte and Doolittle.
13. Following this analysis run a helix prediction using the Chou and Fasman algorithm.
Question 6: Copy and paste the graphs below. You should identify a hydrophobic,
helical region by looking at the two graphs. Enter the numbers of the stretch of amino
acids (i.e. 67 - 91). What is the significance of this hydrophobic helical region?
(4)
Type your answer here
14. Go to http://www.sbc.su.se/~miklos/DAS/
15. Paste the sequence into the query window and run DAS.
Question 7: What does the programme TMDAS do? Read the information on the web
site.
(1)
Type your answer here
Question 8: Copy the DAS curve below. What can you deduce about the protein? (2)
Type your answer here
Question 9: Use ALL the evidence you have gathered in these exercises to write a
paragraph summarising the properties of Godfrey’s protein and suggest a function.
Give a logical step by step explanation. If you make a statement, support it with some
information you have obtained from your database searches.
(3)