Biochemistry of the cell Fall 2013
Bioinformatics in-class project. In a separate text document please respond briefly to each of the following questions.
What is the function of myoglobin (Mb) and why does it have a higher O2 affinity than hemoglobin (Hb; don't spend a lot of time on this question - we'll come back to it). What aspects of Mb structure support this function?
Find the amino acid sequences for pig (Sus scrofa) myoglobin using the NCBI database - select "protein" from the drop-down menu, then enter your search terms.
- Now find closely related amino acid sequences using protein BLAST (BLASTp)
- What animal is most closely-related (excluding results that are either S. scrofa itself, or begin with “Chain A…”)?
- How likely is it that these two sequences are similar due to chance alone (as opposed to sharing a recent common ancestor)?
- Are the sequences exactly the same?
- If not, in what ways do they differ?
Using the NCBI protein data base find myoglobin a.a. sequences for pig, bowhead whale, sperm whale, and Sowerby's beaked whale.
- copy the FASTA version (for a particular protein, see link at top left of page) of these sequences and paste them into a text document (FASTA is described here).
- Use BLASTp to compare just these sequences. Paste the FASTA sequences into the BLASTp window
- Click the little box that says “align two or more sequences”
- How do these sequences differ?
- Here's a table of single-letter a.a. abbreviations; chart of amino acid structures
- Amino acid replacement terminology: "C282Y" means "at a.a. position 282 a cysteine (C) has been replaced by a tyrosine (Y)."
- On the BLAST results page, near the top, you'll see links for "phylogeny" and "multiple sequence alignment" - you may find these useful
- For a.a. at positions 13, 53, 66, 117, 141 & 152: make a simple chart showing these changes for pig --> bowhead whale, bowhead whale --> sperm whale, etc.
- Save the results of this comparison.
Use CN3D to view the structures for sperm whale myoglobin (MMDB ID 41984)
- When you open a structure file in CN3D you should also see the a.a. sequence for that structure in a separate window. If you put the cursor on a particular a.a., in the bottom of that window you can see what number that a.a. is in the sequence. If you click on that a.a., it will be highlighted in the sequence, and in the structure above.
- Do the a.a. differences between related sequences that you observed in the previous section appear to be on the surface of the protein, or in the interior?
- Is there a pattern in how the a.a. changes alter total charge on the protein if we put the organisms in the following order?
- pig, bowhead whale, sperm whale, Sowerby's beaked whale
Do your observations of a.a. replacement and the position of the amino acids in the protein agree with Better oxygen delivery, by E. L. Rezende? Why has myoglobin evolved this way - what's the benefit to the organisms?