bchm628_11_ex4 by panniuniu

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									Exercise 4: PSI-BLAST & PHI-BLAST
Due Date: Friday, June 19 by 12:00 pm.

Name:


4-1)   PSI-BLAST to identify remote homologs

In the Lodge lab, we have identified several proteins that may be putative stress sensors for the
pathogenic fungi, Cryptococcus neoformans. A stress sensor is a protein that detects certain
compounds in the environment and triggers a cellular response; therefore they may have
properties of secondary messengers. One protein that we are looking at has 2 different protein
domains, WSC and DUF1996 domains. The WSC domain has been characterized as a
carbohydrate recognition domain and in the well-studied organism, S. cerevisiae, is found in the
stress-sensor proteins WSC1 and WSC2. The DUF1996 domain is a domain conserved in several
hundred proteins, but has no known function.
        You will use BLASTP and PSI-BLAST to try to determine what taxonomic range of
organisms might have proteins similar to Psi8. A second objective is to determine if there are
any proteins of known function that are remotely similar to Psi8.
     1. Download the Psi8 protein from the exercise 4 homepage.
     2. Conduct a BLASTP search of the NR database using BLOSUM45 as the matrix, max target
         sequences = 500 and default settings for the rest of the parameters.
     3. Repeat steps 1-2 with the Psi8_Nterm protein sequence. This sequence contains the
         first 280 aa, representing the DUF1996 domain.
     4. For both Psi8 and Psi8Nterm, run three iterations of PSI-BLAST against the NR using
         BLOSUM62 as the scoring matrix.
     5. Download the resulting PSSMWithParameter files and save them to your hard drive.
     6. Do a PSI-BLAST of Refseq with each of the PSSM files.
     7. Do a PSI-BLAST of SwissProt with each of the PSSM files.

Data to present in table format:
    List any matches to NON-FUNGAL organisms from the BLASTP of NR using Psi8 and
      Psi8Nterm as the queries. Include accession number, species, query coverage, e-value and
      protein domains found in the sequence from the non-fungal match.
    List the top matches (1 for each search) for the PSI-BLAST of Refseq and SwissProt with
      both PSSM files. Include the Refseq or SwissProt accession number, description, Query
      coverage and E-value.
    Think about how to present this data concisely but in a manner that still allows a reader
      to understand what you’ve done. You will probably want to list the query, blast program
      and database searched in the table as well.

HINT: ascomycetes and basiomycetes are the 2 phylum within the fungal kingdom




BCHM 628 2011                        Exercise 4                                  Page 1 of 3
Questions to consider in the write-up:
1) In the initial BLASTP searches of the NR database, did either of the queries return matches to
       non-fungal organisms?
2) If you found a non-fungal match, what domains were present in the best-scoring non-fungal
       match?
3) Which query gave you more matches to non-fungal proteins? Why do you think that might
       be?
4) In the PSI-BLAST searches of Refseq, did either of the queries using the PSSM files return a
       match to non-fungal organisms? If you found a non-fungal match, what domains were
       present in the best-scoring non-fungal match?
5) Does this analysis give you any more insight into the possible function of the uncharacterized
       DUF1996 domain?
6) Did you find any evidence to support the theory that proteins containing either of domains are
       found in higher (multicellular) eukaryotes?


4-2)   Use of PHI-BLAST and multiple sequence alignment to identify homologs with
       conserved active sites

In exercise 3 you searched for homologs of human chitinase proteins in the Cryptococcus
genome. In this section, you will use a chitinase from human and one from a plant to search
more specifically for active chitinases by including a PROSITE pattern that describes the active
site of two different families of chitinases.
     Do a PHI-BLAST of Refseq limited to Cryptococcus taxon using human CHIT1 as the query
         and including the chitinase family 18 signature pattern
     Do a PHI-BLAST of Refseq limited to fungi using a chitinase from the plant Arabidopsis
         thaliana (NP_566426) as the query and including the chitinase family 19 signature
         pattern
     Do a PHI-BLAST of Refseq with no limitations using the same Arabidopsis chitinase as the
         query and including the chitinase family 19 signature pattern

Questions to consider in the write-up:
   1) Did any of the Cryptococcus matches to human CHIT1 have a match to the chitinase family
      18 active site signature? Where was it located? Did it contain the residue critical for
      activity?
   2) For the best Cryptococcus match to human CHIA, were there any deviations from the
      chitinase family 18 signature pattern? If so, were these deviations conservative changes
      in terms of the physical-chemical properties of the substituted amino acids?
   3) Did you find evidence of any chitinase family 19 proteins in a fungal genome?
   4) What range of taxonomic groups that did have a match to a chitinase family 19 protein?

Data to present in table format:
Top match for each of the PHI-BLAST searches, including Refseq accession number, description,
e-value, position of chitinase signature in the top match and the sequence of the chitinase
signature in the top match.

BCHM 628 2011                        Exercise 4                                  Page 2 of 3
Chitinase family 18 signature:
[LIVMFY]-[DN]-G-[LIVMF]-[DN]-[LIVMF]-[DN]-x-E

Chitinase family 19 signature:
C-x(4,5)-F-Y-[ST]-x(3)-[FY]-[LIVMF]-x-A-x(3)-[YF]-x(2)-F-[GSA]




BCHM 628 2011                          Exercise 4                Page 3 of 3

								
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