You will use information on the World Wide Web to answer the questions below.

EXERCISE 1: USING BIOCHEMISTRY TO DETERMINE PHYLOGENETIC RELATIONSHIPS

To answer these questions, you will use the Structural Classification of Proteins (SCOP) site, a database showing the sequences of different proteins of many different species. For a faster connection, choose the site closest to you from the List of Mirror sites of SCOP. The information at each site is the same, but the connection speed may be faster if you select a site closer to your computer.

Print out the worksheet below and use the link above to answer the following questions.

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Name _____________________________________________________

Link and Think
Using Biochemistry to Determine Phylogenetic Relationships

Use the Structural Classification of Proteins (Choose the mirror site closest to you.) to find the information needed to complete this worksheet. You will also need to refer to the Think About It on page 309 of Biology: The Web of Life.

Partial Sequence Search

After choosing a mirror site, choose Sequence Similarity Search as your access method. Paste in the sequence of the first 25 amino acids in dog cytochrome c protein (GDVEKGKKIFVQKCAQCHTVEKGGK). Do not change any of the default settings (return 20 hits, use the pdb90s sequence library, etc.). Now hit "Run Blast Query." Scan the form returned to you and answer the following questions.

1. Fill in the number of differences between the first 25 amino acids of the cytochrome c of dog and that of horses, tuna, and rice (comparison with rice cuts off after 23 amino acids because the last two amino acids are different). Refer to your textbook for infomation on moth and wheat.

Organism	Horse	Tuna	Moth	Wheat	Rice
Number of differences (with dog)

2. The matching sequence of rice cytochrome c is offset from the sequences of dog, horse, and tuna. How many extra amino acids precede the matching sequence? What is the matching sequence?

3.Fill in the table by comparing the cytochrome c sequences of these species to the corresponding 25 amino acid sequences in other species. (Add "AG" to the rice sequence you found above to extend it to 25 amino acids.)

Organism	Horse	Tuna	Moth	Wheat	Rice
Number of differences with horse
Number of differences with tuna
Number of differences with moth
Number of differences with wheat
Number of differences with rice

4. Based on the biochemical comparisions you made in 3, determine which organism is most closely related to the horse. Is this what you would expect based on physical similarities? Name a physical characteristic that the horse shares with this organism that it does not share with its next closest relative.

Full Sequence Search

Click the full sequences hyperlink to see the full sequences of the top 20 matches. Copy the full sequence of horse cytochrome c . (It is 104 amino acids long and is abbreviated as c1hrc.) Run a new search, just as you did before, using this full sequence.

5. What top three matches does the database return, excluding horse? What is the percentage of identities (matches) of horse to these three organisms? Which two organisms branched off from a common ancestor at about the same time?

6. What is the longest identical sequence for all three organisms? What is the position of the first amino acid in this conserved sequence, as based on the horse sequence? Hint: copy the comparison line (between query and subject lines) and paste them up on top of each othe. Then display them in a fixed width font like Courier.

7. What is cytochrome c? Describe its structure, location, and function after referring to your book and visiting the cytochrome c page. This page describes a particularly important amino acid. What is it and what is its one-letter abbreviation? For a table of amino acid abbreviations, visit Amino Acids.

8. The particularly important amino acid binds to the iron atom (gray ball) of the heme group and helps to hold it in place. It is among the 25 amino acids of cytochrome c compared in the first half of this exercise. What position does it occupy in dog/horse cytochrome c? You can use your book or SCOP to help you find the answer.

9. The iron atom is also bonded to a methionine residue found in the segment you identified in question 6. What is the position of this methionine?

Bonus: You can view and rotate 3D models of cytochrome c, if you install the Chemscape Chime or Rasmol plug-in into your browser. After you have installed one of the plug-ins and restarted your browser, go to Mitochondrial Cytochrome c from Horse. If you are using Rasmol, click the green icon for 1hrc. If you are using Chime, click the red icon.

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Check your work here.

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