Download Preparation and transformation of competent bacteria: Calcium

Bioinformatics and Protein Structure – Questions
Step 1. Getting sequences and learning about your protein
Gene database at NCBI
1. Is this a primary or a secondary database? Explain your answer.
2. What type of information may be available for a gene in this database?
3. What subset of sequences is included in this database?
4. What is a refseq? (You will have to search the NCBI Web Site to find this – go back to the
home page and select NCBI Web Site from the drop down list).
PTGS entry
5. What is the official Gene symbol?
6. What is Gene name?
7. What is the GeneID number?
8. Where in the human genome is this gene located?
9. What is the function of this protein?
10. What specific role does it play in the cell?
11. What is the RefSeq accession number for the mRNA of this gene?
12. What is the RefSeq accession number for the protein sequence (it is the product of the
gene)?
Swiss-Prot
13. Is this a primary or secondary database? Why?
14. Would you expect to see the same protein sequence represented more than once? Why or
why not?
15. What is the SwissProt accession number? (Hint: for proteins it always starts with a P),
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Bioinformatics and Protein Structure – Questions
16. What are the alternate names for this protein?
17. Where in the cell is this protein located? (If it is known it will be in the comments – if it is not
listed your answer is “unknown”.)
18. Does this protein exist as a monomer, dimmer (homo or hetero) etc.?
OMIM
19. What information is available at this site?
20. Is this a primary or a secondary database? Why?
again it is annotated so a 2ndary database
21. What book is the information in OMIM base on?
22. Would you expect to find information on an infectious disease such as Herpes in this
database? Why or why not?
23. What disease is associated with the protein you are studying?
Project specific questions
24. In addition to the general questions, answer the following PAH specific questions (use
information from these databases or the Berg reading material, note that you may need to
clink on links). Please indicate which source each answer comes from.
a. What metabolic pathway does this protein belong to?
b. Which three amino acid residue numbers bind the iron atom and how was this
determined?
c. Which residue is modified by phosphorylation?
d. Diagram the reaction catalyzed by this enzyme (include structures).
e. What is the difference between PKU and the related disease hyperphenyalanine?
f.
Suggest an explanation for why there is a range of symptoms among different
hyperphenylalaninemia individuals (I’m looking for your own ideas here)?
g. Normally, what fraction of phenylalanine is converted to tyrosine?
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Bioinformatics and Protein Structure – Questions
Step 2. Finding related sequences and setting up a multi-sequence FASTA file
BLAST (at NCBI)
25. What does this acronym stand for and what is it used for?
26. In your own words, describe what FASTA format is.
Step 3. Creating the MSA using ClustalW
27. What information can be obtained from a multiple sequence alignment of related proteins?
28. What are three ways this information can be used?
29. What types of sequences can be aligned by ClustalW?
30. Print the output to hand one in at the end of today’s lab. Also answer the following
questions.
31. What is the mutation? Write it in the following format "Res123Res" where Res is the threeletter code for the amino acid in the un-mutated (wild type) protein and the second Res is
the amino acid in the mutated protein. In place of "123" put the amino acid residue number
of the mutation.
32. Is the mutation in a region of conservation – how do you know?
33. What properties differ between the mutant and normal protein (see Betts and Russel,
2003)?
Step 4. Secondary structure prediction
34. What is the secondary structure predicted for the region containing the mutation?
35. What type of secondary structure(s) is the most common in the alignment? If it seems an
even mix between -helixes and -sheets, state that.
36. Do you think that the mutation in your protein may alter the secondary structure, why or why
not?
37. How could you test your prediction?
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Bioinformatics and Protein Structure – Questions
Step 5. Analysis of the 3D structure
38. Experimental method used to obtain the data:
39. Resolution of the structure
40. Species the protein was obtained from
41. List any ligands, cofactors or metal ions included in the structure (particularly important for
enzymes):
Project specific questions. These can be answered using the PDB specific information available
at RCSB, or by going to the journal article on which this structure is based (direct link to the
abstract at RCSB).
a. What is the first author and journal name for the primary citation for this crystal structure?
b. Was the entire PAH protein used to obtain the crystal structure (if not what portion was
included)?
c. What molecules were bound to the protein and what ‘real’ molecules do they represent?
d. Compare and contrast the structure of the substrate analog with phenylalanine.
42. The actual secondary structure of your protein is shown in Control Panel. Using the same
symbols as for the predicted structure, but a different colour pen, write the actual secondary
structure above the predicted secondary structure on your MSA. What major differences, if
any, do you see?
43. PSIPRED states its predictions are ~80% correct. Do you agree this is a good estimate of
the accuracy?
44. What groups, and what exact atom of each group is involved in the H-bond (for example
backbone O of Glu14)?
45. What type of secondary structure are these groups involved in?
46. List any changed, lost, or new H-bonds between the mutated amino acid and other groups.
47. If a new group(s) has been associated with the mutant amino acid (ct the normal amino
acid), what types of structure(s) is this group(s) involved in?
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Bioinformatics and Protein Structure – Questions
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