Globin Sequence Alignment and Evolution

Resources& Websites

• Figure 5-22, Chapter 5
• UniProt (The Universal Protein Resource):
  • http://www.uniprot.org

Exercise

In this exercise, theamino acid sequences of various globin subunits from humans will be retrieved and aligned using tools from UniProt.Protein sequences are typically aligned by comparing amino acid identities, amino acid types (see Chapter 4), amino acid similarities, and protein structural motifs or domains.A set of symbols are typically used to identify identical and similar amino acids in aligned sequences; the UniProt tools use the “*”, “:”, and “.” symbols. The aim of this exercise is to use aligned globin sequences to gaininsight into the evolution of hemoglobin.

Instructions

1. Go to:http://www.uniprot.org.
2. In the search field, enter “human myoglobin” and click on the search button.
3. The following page lists all the UniProt entries that relate to human myoglobin.
4. Choose the entry containing more than 150 amino acids by clicking on the check box on the right side of the entry/identifier number.
5. Once clicked/checked, scroll to the top of the page and click on “Add to basket.”
6. Return to the search field, enter “human hemoglobin,” and click on the search button.
7. The following page lists all the UniProt entries that relate to human hemoglobin.
8. Select/check the entries that relate to the following hemoglobin subunits and contain at least 140 amino acids:
   1. Alpha
   2. Beta
   3. Gamma-1 (fetal beta-like)
   4. Delta (adult beta-like)
   5. Epsilon (embryonic beta-like)
   6. Zeta (embryonic alpha-like)
   7. Mu (adult alpha-like)
   8. Theta-1 (fetal globin)
9. Once all these entries have been selected, click on “Add to basket.”
10. Now go up to the top right corner of the page and click on “Basket.”
11. This opens a small window that lists all the entries in the Basket.
12. Select/check all of the 8 hemoglobin and 1 myoglobinentries.
13. Once complete, click on the “Align” button at the bottom left of the window.
14. After a few moments, the alignment procedure is complete and a page displaying the arrangement of sequences from all of the entries appears.
15. To ensure that all the necessary information is displayed, click/check on the “Tree” and “Result Info” options on the top left side of the page, under the heading “Display.”
16. To ensure that the alignment contains 9 different proteins, scroll down to the bottom of the page and inspect each entry.
17. As appropriate, use the “Annotation” tools on the left side of the page (under the “Highlight” heading) to selectively highlight amino acids with specific properties (e.g., metal binding, aromatic,etc.).
18. Use the combined information on this page to answer the questions below.

Results

1. According to the analysis, how many identical and similar amino acids are fully conserved across myoglobin and the hemoglobin subunits? (Hint: see the information listed in the “Results information” section just below the “Query Sequence” box.)

1. 15 amino acids are conserved by identity, while 38 are conserved by similarity.
2. 38 amino acids are conserved by identity, while 15 are conserved by similarity.

2. Using residue 7 from myoglobin as a reference, which of the following amino acid properties is conserved across all proteins at this position?

1. nonpolarity
2. ability to form a positive charge
3. ability to form a negative charge
4. polarity

3. Which of the following properties best describes the conserved amino acids that are aligned with residue 119 of myoglobin?

1. the ability to form hydrogen bonds
2. the ability to form ion pairs
3. the ability to form positive charges
4. the ability to serve as a base

4. Which of the following properties best describes the conserved amino acids that are aligned with residue 73 of myoglobin?

1. hydrophilic R groups
2. hydrophobic R groups
3. basic R groups
4. acidic R groups

5. By using the Highlight tool for aromatic amino acids, determine which of the following statements is correct.

1. Only W is fully conserved across all proteins at a minimum of 1 position.
2. Only Y and F are fully conserved across all proteins at a minimum of 1 position per amino acid.
3. Only H is fully conserved across all proteins at a minimum of 1 position.
4. Each of the aromatic amino acids is fully conserved across all proteins at a minimum of 1 position per amino acid.

6. The iron atoms of hemoglobin and myoglobin are bound to ___ metal-binding amino acids. These amino acids are __________.

1. 2; fully conservedhistidines
2. 2; histidine and aspartate
3. 18; fully conserved histidines
4. 9; fully conserved histidines

7. According to the sequence comparison tree, myoglobin and hemoglobin are evolutionarily related.The alpha subunit of hemoglobin is most closely related to the ______ subunit.

1. zeta
2. beta
3. mu
4. theta

8. If the length of the connecting lines in the tree is a rough measure of time, which of the following subunits was the last to diverge from the primordial globin?

1. beta and delta
2. gamma-1 and epsilon
3. alpha andtheta
4. mu

9. According to the sequence comparison tree, which of the following subunits was the first to diverge from the primordial globin?

1. zeta
2. beta
3. mu
4. alpha

10. Which of the following statements best describes the evolution of globins?

1. One gene repeatedly mutated over time to ultimately yield multiple forms of the globin protein.
2. The original globin gene was duplicated, and the copy mutated to yield a new globin; this process of gene duplication and mutation occurred repeatedly.