Download Preparation and transformation of competent bacteria: Calcium

BI PAH questions 2009
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Step 1. Getting sequences and learning about your protein
Gene database at NCBI
9. Is this a primary or a secondary database? Explain your answer.
10. What type of information may be available for a gene in this database?
11. What subset of sequences is included in this database?
12. Explain what refseq is. (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
13. What is the official Gene symbol?
14. What is Gene name?
15. What is the GeneID number?
16. Where in the human genome is this gene located?
17. What is the function of this protein?
18. What specific role does it play in the cell?
19. What is the RefSeq accession number for the mRNA of this gene?
20. What is the RefSeq accession number for the protein sequence (it is the product of
the gene)?
21. Is this a primary or secondary database? Why?
22. Would you expect to see the same protein sequence represented more than
once? Why or why not?
23. What is the SwissProt accession number? (Hint: for proteins it always starts with a
P),
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BI PAH questions 2009
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24. What are the alternate names for this protein?
25. 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”.)
26. Does this protein exist as a monomer, dimmer (homo or hetero) etc.?
OMIM
27. What information is available at this site?
28. Is this a primary or a secondary database? Why?
29. What book is the information in OMIM base on?
30. Would you expect to find information on an infectious disease such as Herpes in
this database? Why or why not?
31. What disease is associated with the protein you are studying?
Project specific questions
32. 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?
residue 16 (SwissProt)
d. Diagram the reaction catalyzed by this enzyme (include structures).
e. What is the difference between PKU and the related disease
hyperphenylalaninemia?
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BI PAH questions 2009
f.
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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?
Step 2. Finding related sequences and setting up a multi-sequence FASTA file
BLAST (at NCBI)
33. What does this acronym stand for and what is it used for?
34. (a) from pg 110 In your own words, describe what FASTA format is.
Step 3. Creating the MSA using ClustalW
34. What information can be obtained from a multiple sequence alignment of related
proteins?
35. What are three ways this information can be used?
36. What types of sequences can be aligned by ClustalW?
37. Print the output to hand one in at the end of today’s lab. Also answer the following
questions.
38. What is the mutation? Write it in the following format "Res123Res" where Res is
the three-letter 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.
39. Is the mutation in a region of conservation – how do you know?
40. If the mutation was in a region of conservation what would this suggest?
41. What properties differ between the mutant and normal protein amino acid(see
Betts and Russel, 2003)?
Step 4. Analysis of the 3D structure
42. Experimental method used to obtain the data:
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BI PAH questions 2009
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43. Resolution of the structure
44. Species the protein was obtained from
45. List any ligands, cofactors or metal ions included in the structure (particularly
important for enzymes):
46. 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.
47. The actual secondary structure of your protein is shown in Control Panel. Write the
actual secondary structure above the MSA that was generated in step 3. Use the
symbol h for helix and s for beta sheet (write nothing for loops).
answer on your MSA
48. What groups, and what exact atom of each group is involved in the H-bond (for
example backbone O of Glu14)?
49. What type of secondary structure are these groups involved in?
50. List any changed, lost, or new H-bonds between the variant amino acid and other
groups.
51. If a new group(s) has been associated with the variant amino acid than was
associated with the normal amino acid what types of structure(s) is this group(s)
involved in?
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