Exercise 1: Protein of Unknown Function from Mus musculus
1. Obtain the “Sequence of a protein of unknown function from Mus musculus” in
electronic form from your instructor.
Mus musculus is the genus and species name for the common house mouse, a model
organism studied by many researchers in the biological sciences.
2. Go to the National Center for Biotechnology Information website:
http://www.ncbi.nlm.nih.gov/
Click on “BLAST”, then click on “Protein-protein BLAST (blastp)”. Copy the mouse
protein sequence and paste it into the “Search” box.
From the dropdown menu “Choose database,” select “refseq.” From the dropdown
menu under “Options for advanced blasting,” select “Homo sapiens” so that you will
be searching for human proteins similar to the mouse protein. Be sure that the “Do CDSearch” box is checked. When this boxed is checked, a search for conserved protein
domains will be conducted. Leave all other settings and parameters the same and click
on the BLAST! button.
3. In the next window that appears, there will be a colored, diagrammatic
representation of the mouse protein sequence showing the locations of any
functional/structural domains that are present in the protein. Click on the domain(s) to
find out what they are. Take some time to explore the various links describing the
domain(s). For instance, try clicking the [+] symbols on the left. This should give you
some clues about the identity of the mouse protein.
4. In the original window showing the diagram of domains in the mouse protein, click
the “Format!” button to see the BLAST results for the mouse protein. The BLAST
results will appear in a new window; print this BLAST report. If you scroll down through
the BLAST results, you will see many sequence alignments one after the other. Each
alignment is a sequence comparison between the mouse protein and a human protein
that is similar in sequence to it. The first alignment compares the mouse protein and a
human protein that is the best match to the mouse protein; the second alignment
compares the mouse protein and a human protein that is the second best match to the
mouse protein; and so on…
To infer the function of a protein about which little is known, one can compare the
sequence of the “unknown protein” to other proteins of known function. If the unknown
protein is very similar in sequence to a protein of known function, then there is a good
chance that the unknown protein has the same function as the known protein.
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Use the space provided to answer the following discussion questions:
A. Is there a conserved domain in the mouse protein? Name the domain and discuss
what you learned about it.
B. Which human proteins are similar in sequence to the mouse protein?
C. Speculate about the identity and function of the mouse protein.
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Think back to what you learned about hemoglobin in class; name at least two amino
acid residues that are important to the structure and/or function of hemoglobin and
explain why they are important. (You may use your textbook or notes.)
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Obtain the handout showing a sequence alignment for human -globin, -globin, and
myoglobin from the instructor in order to do the question below. A number of residues
that are important to the structure and/or function of hemoglobin are highlighted on this
alignment. Compare the alignment on the handout to the first alignment on your BLAST
results.
Use the space provided to answer the following discussion questions:
A. Does the mouse protein have all of the highlighted residues that you know to be
important to the structure and/or function of human hemoglobin? Mark/highlight the
locations of these residues in the mouse protein on the printout of your BLAST results.
B. What can you conclude about the importance of these residues to the structure
and/or function of hemoglobin in both organisms?
C. What might happen if one of these residues was replaced by some other residue?
D. Identify a few residues in the mouse protein that are different from those at the
corresponding position in the human globins. Why do you think these residues differ,
and what can you conclude about the importance of these residues to the structure and
function of this protein in both organisms?
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5. Repeat step 2, but this time select “nr” from the dropdown menu “Choose
database.” From the dropdown menu under “Options for advanced blasting,” select
“Takifugu rubripes,” the pufferfish.
Click on “Format!” to see the BLAST results. Compare your BLAST results here to
those from the first BLAST search for human proteins. Print the pufferfish BLAST
results.
Use the space provided to answer the following discussion questions:
A. Is the mouse protein more similar to human -globin or to pufferfish -globin? Is this
what you might have expected before doing the BLAST search? Why?
B. Does pufferfish -globin have all of the residues highlighted on the handout that you
know to be important to the structure and/or function of human -globin? Mark/highlight
the locations of these residues in the pufferfish protein on the printout of your BLAST
results. If any of these residues differ between pufferfish -globin and human -globin,
are the substitutions conservative?
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6. Repeat step 2, but this time select “nr” from the dropdown menu “Choose
database.” From the dropdown menu under “Options for advanced blasting,” select
“Arabidopsis thaliana.” Arabidopsis thaliana is a plant and is a commonly studied model
organism. Click “Format!” to see the BLAST results. Print the Arabidopsis Blast
results.
Use the space provided to answer the following discussion questions:
A. Is it surprising that an Arabidopsis -globin is not among the BLAST hits from this
BLAST search? Why or why not?
B. Name the Arabidopsis protein that is the top hit from the BLAST search. Based on
its degree of similarity to the mouse protein, in terms of total protein sequence length
and percent amino acid identity, do you think it is likely to have the same structure and
function as the mouse protein? Explain your reasoning.
When finished, turn in your three BLAST reports and answers to the discussion
questions.