Extra Problem using the Structural Bioinformatics Tools

  1. Go to the ExPASy site.
  2. Search UNiProtKB database (select from drop down list top of ExPASy page) for the sequence of goose egg lysozyme from the Western graylag goose. This will bring up the UniProt page for this protein sequence.
  1. What is the Primary accession number for this protein sequence?
  2. Save the FASTA format of the sequence to your desktop.
  3. Use the primary sequence tools to determine the pI, absorptivity at 280 nm, and molar mass for this protein.
    - Go to ExPASy site. Click on Resources A to Z. Find and click Compute pI/MW. Paste the sequence into the box.
    - For more information Find ProtParam tool. and Paste the sequence of enter the Accession number.
    --- At what pH will the protein most easily elute from a ion exchange column?
    --- What is the Molecular Mass of the Protein?
    --- What is the extinction coefficient for the protein at 280 nm?
    -------Suppose you were purifying this protein by HPLC and monitoring the fractions eluted by absorption at 280nm. How many mg of this protein would be found in a 5 mL fraction with an absorbance of 0.035 at 280 nm?
  4. Go back to the UniProt page for your goose egg lysozyme. Do a quick BlastP search. 
    At the top of the result page NOTE the various datbases that can be seached via Blastp.
    - In the panel below FILTER the results by limiting the hits to the UniProtKB/SWISS-Prot database
    - Scroll down the page to detailed results.
    - What information is found from a BlastP search?
    - Scroll down until you find the alignment of Western graylag goose with Ostrich lysozyme.
    ---- What is the E-value for the match?
    ---- Note the Primary accession number for Ostrich lysozyme then click on the word align?
    -------- Note the symbols ( *  .  : ) used below the aligment. Based on your knowledge of the amino acids what is the significance of the symbols in the match?
  5. Go back to the UniProt page for your goose egg lysozyme.
    Scroll down to the Features section.
    --- Select HELIX 49-59 by mousing over the secondary structure map and clicking on the blue section cooresponding to that helix.. Note the sequence of this helix.
    --- Draw helical wheel for this helix. You can check with http://trimer.tamu.edu/cgi-bin/wheel/wheel.pl
    Is the helix likely to be completely buried, partially solvent exposed, or completely solvent exposed?
  6. Search the protein data bank for goose lysozyme. OR find the pdb identifier on the UniProtKB page. Note the pdb file name (remember Hen egg lysozyme is 1HEW).
    Go to the CE page and click on TWO CHAINS. Enter the pdb names for hen and goose lysozyme in the appropriate blanks. They are only one chain but enter chain A for each. Click on Calculate Alignment button. Click the -Press to start protein compare- button. What is the P-value, %id, and RMSD (Root mean square deviation) of the backbones for these two structures? Make sure to review the definition of P-value by mousing over the word. Is the match significant?

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