Pabio/Meded 536 Protein BLAST and Motifs – Homework Purpose: Become familiar with Protein BLAST queries of the NCBI non-redundant databases, using the human acyl-CoA binding protein as a query. To examine the range of BLAST hits, we will restrict the output to C. elegans sequences. We will examine the BLAST alignment outputs for a number of the top BLAST hits and relate the alignments to the amino acid conservation that is obvious from multiple alignment of the human, bovine, duck and yeast ACBP family members (see alignment link in question 7). Examine this alignment and identify recognizable conserved motifs which you will use as the basis for determining homology of the C.elegans sequences with the ACBP protein family. Initially you will evaluate the C.elegans hits by eye, looking for conservation of your chosen motifs. Provide your assessment and indicate your reasons. Then you will take each C.elegans sequence you believe has homology to the ACBP family and do a new protein BLAST search against the Conserved Domain database (you can do this analysis as a protein BLAST search against the non-redundant database, since your protein will be automatically searched against the Conserved Domain database – or you can link to the Conserved Domain database and do the search directly from the links at this site.) You will do two different analyses using the information from the Conserved Domain search. First, reevaluate your assessment of the homology of the C. elegans sequences to the ACBP family. Second, determine whether these C.elegans sequences have any other motif recognized by the conserved domain search. You will then use the multiple repetition PSI-BLAST search, which computes an ACBP-specific scoring matrix based on the aligned ACBP sequences, in an attempt to identify additional C.elegans sequences that are distantly related to the ACBP family. Using all this information you will determine whether C.elegans has an ortholog of human ACBP, providing your criteria. In addition, you will determine how many of the C.elegans sequences contain a recognizable ACBP domain. From this make a guess as to the evolution of the C.elegans ACBP family. Finally, you will use PHI-BLAST, to combine a pattern search (A pattern you have determined from the alignment linked in question 7) with BLAST to again search the non-redundant protein database (limited to C.elegans sequences) to evaluate how PHI-BLAST can be used to detect distant similarities. The first extra credit question will test your ability to compare BLAST results for ACBP family members in humans and drosophilia to determine the evolutionary history of ACBP in these organisms and whether they differ from the C.elegans ACBP family members. The second extra credit question will introduce you to phylogenetic analysis. Sequence: human ACBP 87aa form MSQAEFEKAAEEVRHLKTKPSDEEMLFIYGHYKQATVGDINTERPGMLDFTGKAKWDAWNELKGTSKEDAMKAYINK VEELKKKYGI Question # 2 Hyperlink QUESTION # 7 Hyperlink