Lesson Objectives

• How are human proteins similar and different to other primates?
• Can the Differences show evolutionary trends?

Overview of Comparing Primate Proteins

This change has lead us to a great number of questions. When did these changes occur and what is the path that shows our ancestral form’s genealogy? Who were our direct ancestors and what are the side branches? When did this branching occur? Can we learn about the past history of our species by comparing modern organisms to man?

Biologists have begun to use sequences of amino acids in proteins and nucleotide sequences of DNA as an evolutionary clock. Those organisms which have more alterations in their protein sequences are being classified farther away from other organisms in an evolutionary sense when compared to those with more common sequences. In the past Biologists and Paleontologists have used fossils, homologous structures and comparative embryology to help classify organisms and also to try to determine how closely related two organisms actually are.

Scientists have found that certain kinds of protein in different primate species contain many of the same sequences of amino acids (a property called conservation). Also organisms that are similar, seem to have similar biochemistry such as hemoglobin sequence and structure. The similarities of these proteins indicate similarities in the DNA of the organisms. Scientists believe that the more closely two species DNA are, the more closely related they must be. There seems to be a correlation between the number and types of differences and to the phylogenic "closeness" of the organisms compared.

DeVries theory of mutations provided an explanation as to how the variations within a species (and from species to species) could occur. Changes in the sequence of the hereditary material (DNA nucleotides) lead to alterations in the structures and functions of the organism ( or protein). Some changes were bad and lead to disorders or death, some had no effect as the actual protein sequence was left unaltered and a rare few mutations actually made the organism better at competing in their environment.

Those organisms who survive and reproduce are evolutionarily successful. And, those members of the population with better adaptations survive more frequently and pass on those successful traits. Thus the species changes (or evolves) as it becomes more and more like the surviving population. Finally, over time the population may acquire enough differences that it is no longer capable of reproducing with other organisms like the original species type. The history of Homo sapiens has shown this accumulation of differences since it diverged millions of years ago from other primate forms.

Now with modern analysis tools such as BIOLOGY WORKBENCH (http://workbench.sdsc.edu) we can explore these protein sequences and try to make conclusions for ourselves.

Manual Amino Acid Analysis

1. Figure 1 shows the amino acids found in selected sites in hemoglobin of different vertebrates.

Figure 1: Selected amino acid positions in the Hemoglobin of some vertebrates.

2. Count the number of molecules of each amino acid in human hemoglobin. (Don't miss the second section of data). Record these totals in the appropriate column of Data Table 1.

3. Count the number of molecules of each amino acid of other vertebrates hemoglobin. Record these totals in the appropriate columns of Data Table 1.

4. Going from left to right, note the position of each amino acid. Count the numbers of similarities in the amino acid positions in human hemoglobin as compared with the hemoglobin of the other vertebrates in figure 1. Record your observations in Data Table 2.

5. Reexamine figure 1 and count the numbers of differences in the amino acid positions in human hemoglobin as compared with the hemoglobin of the other vertebrates in figure 1. Record your observations in Data Table 2.

Observations:

Data Table 1:

Data Table 2: Similarities and differences in the amino acid sequences of hemoglobin

Analyzing your Observations:

• 1.a. From your observations in data table 2, which primate is most closely related to the human being?
• 1.b. Which primate is most closely related?
• 2.a. From your observations in data table 2, which non-primate is most closely related to the human being?
• 2.b. Which non-primate is most closely related?

Now, let's see how we can use the power of BIOLOGY WORKBENCH to help us do this.

Using Biology Workbench to compare sequences

Myoglobin is found in the muscle tissue of animals and makes for a good comparison. It is smaller than hemoglobin making it easier to count and compare the amino acids. Other molecules that are common to all organisms can allow for exploration of this type which could include plants, fungi, protists and bacteria.

1. Begin by opening your browser to the website at (http://biology.ncsa.uiuc.edu) and register to access the program. Go to SESSION TOOLS to START A NEW SESSION (run), and name it "comparing primate proteins", or something equally intuitive (start new session). Once this has been done, select PROTEIN TOOLS.

In Protein tools we will begin with a NDJINN - MULTIPLE DATABASE SEARCH. This is like a web search engine like YAHOO! or HOTBOT only it looks through protein databases. (perform selected operation)

2. Choose PDBFINDER and PIR 1 by selecting the appropriate check buttons, and type MYOGLOBIN in the first field box. Then click (search).

3. Select the appropriate check buttons for the animals listed in Table 3 from the choices resulting from the search, some 245 sequences. Record their scientific names on Table 3 for later use. Use multiple choice technique here. (Import to workbench).

Table 3: Scientific names of organisms studied in lab.

4. Select all of the organisms by clicking on the boxes to the left of the names. In the window below select CLUSTALW. - (perform selected operation) When new page appears go to the bottom and (submit). This aligns the sequences.

Analysis of Biology Workbench Results

6. Click the box next to the CLUSTALW set. Choose BOXSHADE in the window and then (run). When new page appears go to the bottom and (submit).

7. OOOH! Look at the colors. Those colors have meanings. What do you think the Green Yellow and Purple colors indicate?

8. Completely Conserved and Partially Conserved portions are colored in green and yellow respectively. Purple indicate the next more closely related amino acids. We're interested in the those non-colored (or non-green non-similar) areas. They represent where more of the evolution is occurring. Subtle differences in the species have accumulated due to mutations of the DNA sequence.

9. Complete Table 4 similarly to the way Table 2 was done. Then click (return).

Table 4: Similarities and differences in the amino acid sequences of _________________

Evolutionary Differences

11. A matrix (like a cross-table or multiplication table) will appear at the bottom of the page. The titles are based on the scientific names you recorded earlier. The bigger the value the longer the organism has had to evolve from the organism in that column. Diagonally are a bunch of 0.00000 values. These show how far the animal has evolved from itself. (Pretty tough to do, huh?).

12. Look at the human row.

• What organism is evolutionarily the closest?
• Next closest?
• What is the most distant primate?
• What is the most distant non-primate?
• Now look at the chimp row. Are the numbers the same?

The chimp and gorilla were within 0.00001 units of each other on man's row . What's happened here? Humans and chimps are the same value as before, but look at the chimp-gorilla value. It's twice the size as the human-chimp value! Propose an explanation for what this could mean.

Further Exploration:

1. Compare sequences for MYOGLOBIN of various animals using the same procedures used above (PDBFINDER).

2. Compare sequences for HEMOGLOBIN of various animals using the same procedures used above (PDBFINDER).

3. Use a membrane protein (like PORIN) and compare organisms from the 5 kingdoms evolutionary matrices.