Name__________________

Protein Analysis Lab

Fiocruz

1.  The complete genome of the bacterium Aquifex aeolicus is predicted to encode 1522 proteins.  Of these, 221 are annotated as "hypothetical protein."

 [5] a. What is the accession number of one of these hypothetical proteins, which has a gene name of aq_1554.

 [5] b. Use the COGnitor, at http://www.ncbi.nlm.nih.gov/COG/cog99nitor.html , to compare this protein sequence to the COG database.  What is the number and title of the COG to which this protein sequence belongs?

 [5] c. What is the phylogenetic pattern for this COG?

 hint: the phylogenetic pattern is a list of organisms represented in this COG, listed by single letter abbreviations.

 [5] d. List the full scientific names of the organisms represented in this COG.

 [10] e. This COG represents an enzyme that performs a key step in glycolysis and gluconeogenesis.  One might expect that all complete genomes would encode this enzyme.  Is there another COG made up of only of proteins from genomes that are NOT included in this COG? (Hint: search by phylogenetic pattern)  What is your reasoning? If so, indicate the COG number, the name of the cog, and the phylogenic pattern.

2. Cytochrome c554, a tetra-heme cytochrome from N. europaea, contains four heme-packing motifs.  It's a single chain protein of two domains.  Its PDB accession is 1BVB.

 [6] a. What is the gi accession for this protein?

 [7] b.  Do a PSI-BLAST search of the sequence again NR. Look at the hit list above and below the threshold. There is a single hit with a PDB accession.  What is the PDB accession of this hit?

[7] c.  Click the check box next to the hit in b and do a single iteration of PSI-BLAST.  What is the e-value between 1BVB and this hit?

[15] d.  Is 1BVB and this hit VAST neighbors of each other?  If yes, are the PSI-BLAST alignments and the structure alignments similar?  If not, explain why two proteins are PSI-BLAST neighbors and not VAST neighbors of each other..

3. The iron responsive element binding protein 2 is a large single chain protein that binds to iron responsive element RNA hairpin loop in the 5' UTR of ferritin mRNA.  IRE protein also has a second function.

 [5] a. Do a BLAST search on the IRP2, gi 1352477 to determine the second function? Hint: look at the definition lines of the BLAST hits.

[5] b. Find a structure that is associated with this second function. Look for the structure summary page for this structure in MMDB. Examine it in Cn3D. How many domains does this structure have?

[13] c. Now look at the structure of the IRE RNA, pdb accession 1AQO. From your observation of the structure from part b, can IRE protein bind to the IRE RNA and perform its other function simultaneously?  Explain why or why not.  Hint: Iron binding site is required for the activity of the protein in b). Thus, using genbank records for IRP2 and the structure in b) determine if the two functions can occur simultaneously.

[12] d. Arginines are important residues in RNA binding. Can you identify conserved arginines between human IRP1(gi 284275), another IRE binding protein, and the structure from Bos taurus by looking at the BLAST alignment in the sequence window of Cn3D.  Print the alignment and highlight the conserved arginines or write down the positions on IRP1 of the conserved arginines.

(Do not forget to turn-on network access for Cn3D .. under option->net configure and quit cn3d)