Week 8 – Multiple Alignment

1. Perform a multiple alignment using the ACBP liver forms and the ACBP brain forms from this week’s sequence file and show the alignment (ALN file) you obtained (5 pts).

2. Compare the pattern of identical residues conserved within the liver forms to those conserved within the brain forms. Are there brain or liver specific residues (i.e. residues conserved in liver but not in brain or vice versa)? Provide a couple of examples (ie brain: A26, liver: W32) (10 pts).

3. Determine a “pattern” of liver-specific amino acid residues, i.e. those amino acids conserved in all liver forms but not in the brain forms. Provide your pattern. (10 pts) a. Do a PHI-BLAST search using the human ACBP Liver form in the query and your Liverspecific pattern – limit your output to salmonids. Did you detect a putative liver ACBP form in the salmonid “ atlantic salmon salmo” ? Provide the BLAST hit. If you obtain no putative salmon ACBP hits try altering your pattern slightly and redo the PHI-BLAST. If all else fails, do a normal BLAST search with no pattern and exam the hits to see if you can detect a putative liver ACBP form in the salmonid “atlantic salmon salmo”? Provide the BLAST hit. In this last case, how did the salmo sequence differ from your pattern (10 pts) b. Do a multiple alignment of all of the liver forms in question 2 plus your putative salmon liver ACBP form – provide the alignment (10 pts). c. Discuss the alignment with regard to other liver-specific residues that you did not include in your PHI-BLAST pattern, i.e. are those conserved as well? Would you consider this putative atlantic salmon ACBP to be a liver form? Discuss (10 pts).

4. Determine a “pattern” of amino acid residues that are conserved in the ACBP brain forms but not in the liver forms. Provide the pattern (10 pts) a. Do a PHI-BLAST search using the human ACBP Brain form in the query and your Brain-specific pattern – limit your output to salmonids. Did you detect a putative brain ACBP form in the Atlantic salmon (Salmo salar)? If you obtain no putative salmon ACBP hits try altering your pattern slightly and redo the PHI-BLAST. If you still get no putative salmon ACBP hit try doing a protein BLAST without the pattern and look at the hits by eye to determine whether there is a brain form in Salmo salar (hint- you should detect one) (10 pts) b. Do a multiple alignment of all of the brain forms in question 2 plus your putative salmon brain ACBP form – provide the alignment. (10 pts) c. Discuss the alignment with regard to the brain-specific residues? Would you consider this salmon ACBP to be a brain form? Discuss in relationship to your PHI-BLAST pattern and other “brain form” specific characteristics. (10 pts)

5. Do an alignment of all of the liver and brain form ACBP sequences including the salmon sequences. Identify the identical residues conserved within all the sequences and discuss with regard to conservation of the previously characterized “ACBP” motifs (10 points). Can you make any conclusion regarding how the brain and the liver forms differ from each other – relying upon what you know of the structure of ACBP? (FYI..no right answers here) (5 pts)