Think about what determines the structure of a protein. Chemical bonds between the amino acids do. For example, an amino acid with a plus charge, like arginine, might bond with a negative amino acid, like glutamate, to form an ionic bond in the protein that helps keep it together.

The "isoelectric point" of a protein is a number between 0 and 14 that measures the charges on the protein. A higher isoelectric point means there are more positive charges on the protein (or fewer negative charges), and a lower the isoelectric point means there are fewer positive charges (and more negative ones). In this section we'll use a tool that predicts the isoelectric points of our proteins to see if there are changes in charge that might explain the change in structure.

1. Go back to the Protein Tools homepage. You can do this by scrolling to the bottom of the screen and clicking on "Return":



2. Select the tool choice that says "PI -- Isoelectric Point Determination." Unfortunately, we can only run this tool on one protein at a time, so uncheck the mutant protein (1LE2) check box so we calculate the isoelectric point for the wild-type protein (1LPE) first. Then press the "Run" button:



3. On the next screen just press the "Submit" button:



4. On the results page, you will see a table of "pH" and "charge." The isoelectric point is the point where the protein has a net charge of zero. Scroll down to the bottom to find the isoelectric point, as shown below:

5. Then go back to the Protein Tools menu using the "Return" button at the bottom:



6. Next run the same experiment on the mutant protein. Uncheck the "1LPE" box and check the "1LE2" box, then run the program as before.



The number you get for the isoelectric point should be different and lower. This means that the mutant protein has lost some positive charge. This positive charge, on the arginine amino acid that was replaced by cysteine in the mutant, must be important for the structure and function of the protein!

CONCLUSION
Why doesn't the mutant protein work right? We now have some important information, which we found using bioinformatics tools. The change in charge in the mutant apolipoprotein can be seen in the different isoelectric point of the mutant. We also saw using GOR4 that there is a small conformational change in the mutant protein. Remember that we couldn't see that change very well just looking at a picture of the two proteins. This conformational change turns out to be responsible for this form of the disease hypercholesterolemia. The changed shape in the mutant lipoprotein does not allow it to attach onto liver cells which would normally "eat" the cholesterol the lipoprotein delivers. So, instead, the cholesterol cannot be used up by these liver cells and remains in the bloodstream, which causes symptoms and threats such as those mentioned in the beginning of the tutorial.

2. Select BLASTP.

3. In the next window, select the GBBCT database and 250 in the "1-line descriptions" pull-down menu. Click "Submit".


4. To generate a good phylogenetic tree, we need to include organisms that represent all of the different divisions or classes of bacteria. From the search results, select the following sequences:

Alpha, Rickettsia conorii 15619100 Alpha, Rickettsia prowazekii 3860572 Alpha, Agrobacterium tumefaciens 15157847 Alpha, Mesorhizobium loti 14024067 Gamma, Escherichia Coli 42926 Gamma, Vibrio Cholera 3341643 Gamma, Haemophilus influenza 1573838 Beta, Neisseria Gonorrhea 150366 Archae, Methanobacterium thermoautotrophicum 2894537 Epsilon, Helicobacter Pylori 2313275 Bacillus, Staphylococcus Aureus 14246761 Beta, Neisseria meningitidis 7379742 Alpha, Paracoccus denitrificans 975315 Bacillus, Bacillus subtilis 142966 Archae, Archaeoglobus fulgidus 2689375 Archae, Thermoplasma volcanium 14325503

5. "Import" all the sequences selected.

6. These sequences will be listed on the Protein Tools homepage.


7. Select all of the sequences that you just imported, plus the R. americana succinate deH subunit 2 sequence.

8. Select CLUSTALW in the tool menu and click on "Run". Click "Submit" on the next page.

9. The results will be very difficult to interpret at this point, so just click on "Import Alignment." You are now in the Alignment Tools domain of the Workbench.

10. Select the aligned sequences as shown in the image below.

11. Select DRAWGRAM from the tool menu, hit "Run" and click "Submit" on the next page. The phylogenetic tree that you obtain should look something like this:


In order to make sense of this tree, all of the ID numbers need to be substituted with actual bacterial names and the division each bacterium belongs to! The Biology Workbench does not provide a tool to do that, but for the tree above this was done "manually", with the following results:



Now it is easy to see the different divisions (and, hence, groupings) of bacteria. Most importantly, at the very top, notice that R. americana places with the alpha-Proteobacteria, once again indicating the very strong relatedness between them.


A Phylogenetic Tree of the Chloroplast Ribosomal Protein S12 Gene
To construct our second tree, once again we will use a gene that you are already familiar with: the chloroplast S12 ribosomal gene of P. purpurea. As with the succinate deH gene, this gene is present in all organisms and it is also highly conserved,

1. Select the "Chloroplast 30S ribosomal protein S12" sequence.

2. Select BLASTP

3. In the next window, select the GBBCT database and 250 in the "1-line descriptions" pull-down menu. Click "Submit"



4. To generate a good phylogenetic tree, we need to include organisms that represent all of the different divisions or classes of bacteria. From the search results, select the following sequences:

Cyanobacterium, A. nidulans, 11204 Cyanobacterium, Synechocystis, 1653348 Cyanobacterium, Spirulina, 47447 Bacillus, Clostridium, 15026202 Epsilon, Helicobacter, 4155706 Bacillus, Streptococcus, 11493923 Bacillus, Subtilis, 2632267 Bacillus, Aureus, 14246074 Beta, Corrodens, 41653 Chlamydia, Chlamydia, 3328863 Mycobacterium, Leprae, 725269 Mycobacterium, Smegmatis, 511125

5. "Import" all the sequences selected.

6. All of these sequences will be listed on the Protein Tools homepage.



7. Select all of the sequences that you just imported, plus the P. purpurea chloroplast S12 ribosomal gene sequence.

8. Select CLUSTALW in the tool menu and click on "Run". Click "Submit" on the next page.

9. The results will be very difficult to interpret at this point, so just click on "Import Alignment." You are now in the Alignment Tools domain of the Workbench.

10. Select DRAWGRAM from the tool menu, hit "Run" and click "Submit" on the next page. The phylogenetic tree that you obtain should look something like the one shown below:



When the bacterial ID's are substituted with actual names of bacteria and their divisions, we see a clear pattern:



Here we see that the chloroplast S12 ribosomal gene of the simple eukaryote Porphyra purpurea is grouped with the Cyanobacteria, indicating a strong evolutionary relatedness between them.

Conclusion
Using various bioinformatics tools in the Biology Workbench, you have shown that mitochondrial and chloroplast gene sequences from a basic eukaryotic cell are highly homologous to gene sequences in certain groups of bacteria. This type of evidence strongly supports the endosymbiotic theory. The endosymbiotic theory proposes that eukaryotic cells originated from prokaryotic cells that engulfed fellow prokaryotes, and instead of digesting them, formed a symbiotic relationship where one provided the nutrients and shelter, while the other produced more than enough energy for both of them. Specifically, you have found evidence suggesting that mitochondria evolved from alpha-Proteobacteria, while chloroplasts evolved from Cyanobacteria.